U.S. patent number 8,815,821 [Application Number 13/277,957] was granted by the patent office on 2014-08-26 for double-stranded oligonucleotides.
This patent grant is currently assigned to Life Technologies Corporation. The grantee listed for this patent is Tod Woolf. Invention is credited to Tod Woolf.
United States Patent |
8,815,821 |
Woolf |
August 26, 2014 |
Double-stranded oligonucleotides
Abstract
Antisense sequences, including duplex RNAi compositions, which
possess improved properties over those taught in the prior art are
disclosed. The invention provides optimized antisense oligomer
compositions and method for making and using the both in in vitro
systems and therapeutically. The invention also provides methods of
making and using the improved antisense oligomer compositions.
Inventors: |
Woolf; Tod (Natic, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Woolf; Tod |
Natic |
MA |
US |
|
|
Assignee: |
Life Technologies Corporation
(Carlsbad, CA)
|
Family
ID: |
46321777 |
Appl.
No.: |
13/277,957 |
Filed: |
October 20, 2011 |
Prior Publication Data
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Document
Identifier |
Publication Date |
|
US 20130045520 A1 |
Feb 21, 2013 |
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Related U.S. Patent Documents
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Application
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Patent Number |
Issue Date |
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12062380 |
Apr 3, 2008 |
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11049636 |
Feb 2, 2005 |
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10357529 |
Feb 3, 2003 |
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10357826 |
Feb 3, 2003 |
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60615408 |
Sep 30, 2004 |
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60540552 |
Feb 2, 2004 |
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60353203 |
Feb 1, 2002 |
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60436238 |
Dec 23, 2002 |
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60438608 |
Jan 7, 2003 |
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60353381 |
Feb 1, 2002 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
A61P
1/04 (20180101); A61P 27/02 (20180101); C12Y
207/11022 (20130101); A61P 35/00 (20180101); A61P
31/18 (20180101); C12N 15/1137 (20130101); C12N
15/1135 (20130101); A61P 31/12 (20180101); A61K
31/713 (20130101); C12N 15/113 (20130101); C12N
15/111 (20130101); A61P 17/06 (20180101); A61P
43/00 (20180101); C12Y 301/03048 (20130101); A61P
37/02 (20180101); A61P 29/00 (20180101); C12N
13/00 (20130101); A61P 9/00 (20180101); A61P
31/20 (20180101); C12N 2310/11 (20130101); C12N
2310/321 (20130101); C12N 2310/53 (20130101); C12N
2320/50 (20130101); C12N 2310/33 (20130101); C12N
2310/14 (20130101); C12N 2310/315 (20130101); C12N
2320/51 (20130101); C12N 2310/351 (20130101); C12N
2320/31 (20130101) |
Current International
Class: |
C12N
15/11 (20060101) |
Field of
Search: |
;514/44A |
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Primary Examiner: Whiteman; Brian
Parent Case Text
RELATED APPLICATIONS
This application is a continuation of U.S. Ser. No. 12/062,380,
filed on Apr. 3, 2008, now abandoned, which is a continuation
application of U.S. Ser. No. 11/049,636, filed on Feb. 2, 2005, now
abandoned, which claims the benefit of U.S. Provisional Application
No. 60/615,408, filed on Sep. 30, 2004, and 60/540,552, filed on
Feb. 2, 2004. U.S. Ser. No. 11/049,636 is also a
continuation-in-part of U.S. Ser. No. 10/357,529, filed Feb. 3,
2003, now abandoned, and Ser. No. 10/357,826, filed Feb. 3, 2003,
now abandoned, both of which claim the benefit of U.S. Provisional
Application No. 60/353,203, filed on Feb. 1, 2002, 60/436,238,
filed Dec. 23, 2002, 60/438,608, filed Jan. 7, 2003, and
60/353,381, filed Feb. 1, 2002. The entire contents of the
aforementioned applications are hereby expressly incorporated
herein by reference.
REFERENCE TO BIOLOGICAL SEQUENCE DISCLOSURE
This application contains nucleotide sequence and/or amino acid
sequence disclosure in computer readable form and a written
sequence listing, the entire contents of both of which are
expressly incorporated by reference in their entirety as though
fully set forth herein.
Claims
The invention claimed is:
1. A method of decreasing expression of a target gene in a cell,
comprising: contacting the cell with a composition comprising a
double-stranded oligonucleotide, the double-stranded
oligonucleotide comprising: a first nucleic acid molecule having a
first nucleotide sequence that is antisense to the target gene and
having 2'-O-methyl RNA arms and an unmodified DNA gap, and a second
nucleic acid molecule having a nucleotide sequence that hybridizes
to the first nucleotide sequence, wherein the first and second
molecules are each 25 nucleosides in length and are 100%
complementary to each other, wherein the double-stranded
oligonucleotide has no overhanging single-stranded sequence at
either end of the molecule, to thereby decrease expression of the
target gene in the cell.
2. A method of decreasing expression of a target gene in a cell,
comprising: contacting the cell with a composition comprising a
double-stranded oligonucleotide in the presence of a transfection
reagent wherein the transfection reagent is a cationic lipid, the
double-stranded oligonucleotide comprising: a first nucleic acid
molecule having a first nucleotide sequence that is antisense to
the target gene and having 2'-O-methyl RNA arms and an unmodified
DNA gap, and a second nucleic acid molecule having a nucleotide
sequence that hybridizes to the first nucleotide sequence, wherein
the first and second molecules are each 25 nucleosides in length
and are 100% complementary to each other, wherein the
double-stranded oligonucleotide has no overhanging single-stranded
sequence at either end of the molecule, to thereby decrease
expression of the target gene in the cell.
3. A method of decreasing expression of a target gene in a cell,
comprising: contacting the cell with a composition comprising a
double-stranded oligonucleotide in the presence of a transfection
reagent wherein the transfection reagent is a cationic lipid, the
double-stranded oligonucleotide comprising: a first nucleic acid
molecule having a first nucleotide sequence that is antisense to
the target gene and having 2'-O-methyl RNA arms and an unmodified
DNA gap, and a second nucleic acid molecule having a nucleotide
sequence that hybridizes to the first nucleotide sequence, wherein
the first and second molecules are each 25 nucleosides in length to
thereby decrease expression of the target gene in the cell.
Description
BACKGROUND OF THE INVENTION
Complementary oligonucleotide sequences are promising therapeutic
agents and useful research tools in elucidating gene function.
However, oligonucleotide molecules of the prior art are often
subject to nuclease degradation when applied to biological systems.
Therefore, it is often difficult to achieve efficient inhibition of
gene expression (including protein synthesis) using such
compositions.
In order to maximize the usefulness, such as the potential
therapeutic activity and in vitro utility, of oligonucleotides that
are complementary to other sequences of interest, it would be of
great benefit to improve upon the prior art oligonucleotides by
designing improved oligonucleotides having increased stability both
against serum nucleases and cellular nucleases and nucleases found
in other bodily fluids.
SUMMARY OF THE INVENTION
The instant invention is based, at least in part, on the discovery
that double-stranded oligonucleotides comprising an antisense
oligonucleotide and a protector oligonucleotide, are capable of
inhibiting gene function. Thus, the invention improves the prior
art antisense sequences, inter alia, by providing oligonucleotides
which are resistant to degradation by cellular nucleases.
Accordingly, the invention provides optimized oligonucleotide
compositions and methods for making and using both in in vitro, and
in vivo systems, e.g., therapeutically.
In one aspect, the invention pertains to a double-stranded
oligonucleotide composition having the structure depicted in FIG.
1, where (1) N is a nucleomonomer in complementary oligonucleotide
strands of equal length and where the sequence of Ns corresponds to
a target gene sequence and (2) X and Y are each independently
selected from a group consisting of nothing; from about 1 to about
20 nucleotides of 5' overhang; from about 1 to about 20 nucleotides
of 3' overhang; and a loop structure consisting from about 4 to
about 20 nucleomonomers, where the nucleomonomers are selected from
the group consisting of G and A. The invention further includes
compositions such as reaction mixtures comprising such
double-stranded oligonucleotides, as well as methods for using such
double-stranded oligonucleotides.
An "overhang" is a relatively short single-stranded nucleotide
sequence on the 5'- or 3'-hydroxyl end of a double-stranded
oligonucleotide molecule (also referred to as an "extension,"
"protruding end," or "sticky end").
In one embodiment, the number of Ns in each strand of the duplex is
between about 12 and about 50. In other embodiments, the number of
Ns in each strand of the duplex is between about 12 and about 40
(i.e., in the structure above, oligo(N) has between about 12 and
about 50 nucleomonomers); or between about 15 and about 35; or more
particularly between about 20 and about 30; or even between about
21 and about 25.
In one embodiment, X is a sequence of about 4 to about 20
nucleomonomers which form a loop, wherein the nucleomonomers are
selected from the group consisting of G and A.
In one embodiment, two of the Ns are unlinked, i.e., there is no
phosphodiester bond between the two nucleomonomers. In one
embodiment, the unlinked Ns are not in the antisense sequence.
In one embodiment, the nucleotide sequence of the loop is GAAA.
In another aspect, the invention pertains to a double-stranded
oligonucleotide composition having the structure depicted in FIG.
2: where (1) N is a nucleomonomer in complementary oligonucleotide
strands of equal length where the sequence of Ns corresponds to a
target gene sequence; and (2) Z is a nucleomonomer in complementary
oligonucleotide strands of between about 2 and about 8
nucleomonomers in length and where the sequence of Z optionally
corresponds to the target sequence; and (3) where M is a
nucleomonomer in complementary oligonucleotide strands of between
about 2 and about 8 nucleomonomers in length and where the sequence
of Ms optionally corresponds to the target sequence. Although the
sequences of N nucleomonomers should be of the same length, the
sequences of Z and M nucleomonomers may optionally be of the same
length. The invention further includes compositions such as
reaction mixtures comprising such double-stranded oligonucleotides,
as well as methods for using such double-stranded
oligonucleotides.
In one embodiment, Z and M are nucleomonomers selected from the
group consisting of C and G.
In one embodiment, the sequence of Z or M is CC, GG, CG, GC, CCC,
GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC,
CCCG, CGGG, GCCC, GGCC, or CCGG.
In another aspect, the invention pertains to a double-stranded
oligonucleotide composition having the structure depicted in FIG.
3, where (1) N is a nucleomonomer in complementary oligonucleotide
strands of equal length and where the sequence of Ns corresponds to
a target gene sequence and (2) X is selected from the group
consisting of nothing; 1-20 nucleotides of 5' overhang; 1-20
nucleotides of 3' overhang. The invention further includes
compositions such as reaction mixtures comprising such
double-stranded oligonucleotides, as well as methods for using such
double-stranded oligonucleotides.
In some embodiments, X is a loop structure consisting of from about
4 to about 20 nucleomonomers, where the nucleomonomers are selected
from the group consisting of G and A.
In the structure above, M is a nucleomonomer in complementary
oligonucleotide strands of between about 2 and about 8
nucleomonomers in length which optionally correspond to the target
sequence. In one embodiment, M is a nucleomonomer selected from the
group consisting of contain C and G.
In one embodiment, the sequence of M is CC, GG, CG, GC, CCC, GGG,
CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG,
CGGG, GCCC, GGCC, or CCGG.
In another aspect, the invention pertains to a double-stranded
oligonucleotide composition having the structure depicted in FIG.
4, where (1) N is a nucleomonomer in complementary oligonucleotide
strands of equal length and which correspond to a target gene
sequence and (2) Y is selected from the group consisting of
nothing; 1-20 nucleotides of 5' overhang; 1-20 nucleotides of 3'
overhang; a loop consisting of a sequence of from about 4 to about
20 nucleomonomers, where the nucleomonomers are all either G's or
A's and (3) where Z is a nucleomonomer in complementary
oligonucleotide strands of between about 2 and about 8
nucleomonomers in length and which comprise a sequence which can
optionally correspond to the target sequence. The invention further
includes compositions such as reaction mixtures comprising such
double-stranded oligonucleotides, as well as methods for using such
double-stranded oligonucleotides.
In one embodiment, Zs are nucleomonomers selected from the group
consisting of C and G.
In one embodiment, the sequence of Z is CC, GG, CG, GC, CCC, GGG,
CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG,
CGGG, GCCC, GGCC, or CCGG.
In another aspect, the invention pertains to a method of regulating
gene expression in a cell, comprising forming a double-stranded
oligonucleotide composition as described herein and contacting a
cell with the double-stranded duplex, to thereby regulate gene
expression in a cell.
In one embodiment, the invention pertains to a method of increasing
the nuclease resistance of an antisense sequence, comprising
forming a double-stranded oligonucleotide composition as described
herein, such that a double-stranded duplex is formed, wherein the
nuclease resistance of the antisense sequence is increased compared
to a double-stranded, unmodified RNA molecule.
In other embodiments, the invention includes methods for
introducing one or more double-stranded nucleic acid molecule into
cells (e.g., eukaryotic cells). In particular embodiments, such
methods include those comprising contacting cells (e.g., eukaryotic
cells) with one or more double-stranded nucleic acid molecule. In
more specific embodiments, at least the first two (e.g., the first
two, the first three, the first four, the first five, etc.)
nucleotides at the 5' terminus of the first strand of the one or
more double-stranded nucleic acid molecule are chemically modified
at the 2' positions and/or at least the first two nucleotides at
the 5' terminus of the second strand of the one or more
double-stranded nucleic acid molecule are chemically modified at
the 2' positions. In additional specific embodiments, the
double-stranded nucleic acid molecule may be between 18 and 30,
between 20 and 30, or between 22 and 30 nucleosides in length.
Further, the double-stranded nucleic acid molecule may be 25
nucleosides in length. The invention further includes compositions
(e.g., double-stranded nucleic acid molecule, reaction mixtures,
etc.) used in these methods.
In various embodiments of the invention (e.g., those described in
the preceding paragraph), the double-stranded nucleic acid molecule
may contain an overhang (e.g., a 3' overhang and/or a 5' overhang)
of at least one (e.g., one, two, three, four, five, etc.)
nucleoside on at least one end (e.g., one end or both ends).
Additionally, the nucleosides of the overhang(s) may be deoxy
nucleosides such as deoxy-T. In specific embodiments, the
overhang(s) may be or contain deoxy T-deoxy T.
Further, when nucleic acid molecules in compositions of the
invention or used in methods of the invention are chemically
modified at one or more 2' position, the 2' chemical
modification(s) may be a 2'-O-methyl modification, a 2'-O-propyl
modification, a 2'-O-ethyl modification, a 2'-fluoro modification,
or a combination of such modifications. Additionally, 2' chemical
modification on such nucleic acid molecules may be located on
ribose sugars, deoxyribose sugars, or a combination of these
sugars.
Further, nucleic acid molecules of the invention may be combined
with one or more transfection reagent to form a composition.
Additionally, such compositions may be contacted with cells (e.g.,
eukaryotic cells). Transfection reagents used in such methods and
compositions may comprise one or more cationic lipid. One example
of a transfection reagent which may be used in the practice of the
invention is LIPOFECTAMINE 2000.TM.. The invention thus includes
methods which employ and compositions which contain transfection
reagents.
The invention additionally includes methods by which nucleic acid
molecules (e.g., double-stranded nucleic acid molecules) may be
introduced into eukaryotic cells by electroporation.
Further, when double-stranded nucleic acid molecules (e.g.,
double-stranded RNA molecules) are used in the practice of the
invention, one strand of these double-stranded nucleic acid
molecules may be complementary to all or part of the nucleotide
sequence of an RNA (e.g., an mRNA) which is expressed in a cell
(e.g., a eukaryotic cell into which the double-stranded nucleic
acid molecules are introduced). When the RNA is a mRNA,
introduction of double-stranded nucleic acid molecules in cells may
inhibit expression of protein from such RNA. Thus, the invention
includes methods for inhibiting gene expression.
Methods of stabilizing oligonucleotides, particularly antisense
oligonucleotides, by formation of a oligonucleotide compositions
comprising at least 3 different oligonucleotides, are disclosed in
co-pending application U.S. application Ser. No. 10/357,826. This
application and all of its teachings is hereby expressly
incorporated herein by reference in its entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 A depiction of an exemplary composition.
FIG. 2 A depiction of an exemplary composition.
FIG. 3 A depiction of an exemplary composition.
FIG. 4 A depiction of an exemplary composition.
FIG. 5 A depiction of an exemplary composition. (SEQ ID NO:1)
FIG. 6 Shows an exemplary high affinity nucleomonomer.
FIG. 7 A depiction of an exemplary composition.
FIG. 8 A depiction of an exemplary composition.
FIG. 9 A depiction of an exemplary composition. (SEQ ID NO:13)
FIG. 10 A depiction of an exemplary composition.
FIG. 11 A depiction of an exemplary composition.
FIG. 12 A depiction of an exemplary composition.
FIG. 13 A depiction of an exemplary 2 subunit morpholino
oligonucleotide.
FIG. 14A shows that the length of double-stranded oligonucleotides
and the presence or absence of overhangs has no effect on
function.
FIG. 14B shows the effect of structural changes on the efficacy of
siRNAs targeting .beta.-3-Integrin.
FIG. 15A shows that there is no correlation was observed between
the length of the double-stranded oligonucleotide and the level of
PKR induction for the given sequences.
FIG. 15B shows effect of .beta.-3-Integrin targeted 21-mers and
27-mers on PKR expression in HMVEC Cells.
FIG. 16 shows the effect of 5' or 3' modification on activity of
double-stranded RNA duplexes.
FIG. 17 shows the effect of the size of the modifying group on
activity of the double-stranded RNA duplex.
FIG. 18 shows the results of 2'-O-methyl (also referred to herein
as "2'-O-Me") modifications on the activity of double-stranded RNA
duplexes.
FIG. 19 shows the inhibition of p53 by 32- and 37-mer blunt-end
siRNAs.
FIG. 20 provides a schematic representation of a system for
providing a product to a party such as a customer/purchaser.
FIG. 21 provides a schematic representation of a system for
advising a party as to the availability of a product.
DETAILED DESCRIPTION OF THE INVENTION
The instant invention advances the prior art by providing
double-stranded oligonucleotide compositions for use, both in vitro
and in vivo, e.g., therapeutically, and by providing methods of
making and using the double-stranded antisense oligomer
compositions.
Double-Stranded Oligonucleotide Compositions
Double-stranded oligonucleotides of the invention are capable of
inhibiting the synthesis of a target protein, which is encoded by a
target gene. The target gene can be endogenous or exogenous (e.g.,
introduced into a cell by a virus or using recombinant DNA
technology) to a cell. As used herein, the term "target gene"
includes polynucleotides comprising a region that encodes a
polypeptide or polynucleotide region that regulates replication,
transcription, translation, or other process important in
expression of the target protein; or a polynucleotide comprising a
region that encodes the target polypeptide and a region that
regulates expression of the target polypeptide; or non-coding
regions such as the 5' or 3' UTR or introns. Accordingly, the term
"target gene" as used herein may refer to, for example, an mRNA
molecule produced by transcription from a gene of interest.
Furthermore, the term "correspond," as in "an oligomer corresponds
to a target gene sequence," means that the two sequences are
complementary or homologous or bear such other biologically
rational relationship to each other (e.g., based on the sequence of
nucleomonomers and their base-pairing properties).
The "target gene" to which an RNA molecule of the invention is
directed may be associated with a pathological condition. For
example, the gene may be a pathogen-associated gene, e.g., a viral
gene, a tumor-associated gene, or an autoimmune disease-associated
gene. The target gene may also be a heterologous gene expressed in
a recombinant cell or a genetically altered organism. By
determining or modulating (e.g., inhibiting) the function of such a
gene, valuable information and therapeutic benefits in medicine,
veterinary medicine, and biology may be obtained.
The term "oligonucleotide" includes two or more nucleomonomers
covalently coupled to each other by linkages (e.g.,
phosphodiesters) or substitute linkages. In one embodiment, it may
be desirable to use a single-stranded nucleic acid molecule which
forms a duplex structure (e.g., as described in more detail below).
For example, in one embodiment, the oligonucleotide can include a
nick in either the sense of the antisense sequence.
The term "antisense" refers to a nucleotide sequence that is
inverted relative to its normal orientation for transcription and
so expresses an RNA transcript that is complementary to a target
gene mRNA molecule expressed within the host cell (e.g., it can
hybridize to the target gene mRNA molecule through Watson-Crick
base pairing). An antisense strand may be constructed in a number
of different ways, provided that it is capable of interfering with
the expression of a target gene. For example, the antisense strand
can be constructed by inverting the coding region (or a portion
thereof) of the target gene relative to its normal orientation for
transcription to allow the transcription of its complement, (e.g.,
RNAs encoded by the antisense and sense gene may be complementary).
Furthermore, the antisense oligonucleotide strand need not have the
same intron or exon pattern as the target gene, and noncoding
segments of the target gene may be equally effective in achieving
antisense suppression of target gene expression as coding
segments.
Accordingly, one aspect of the invention is a method of inhibiting
the activity of a target gene by introducing an RNAi, also referred
to as RNA interference, agent into a cell, such that the dsRNA
component of the RNAi agent is targeted to the gene. In one
embodiment, an RNA oligonucleotide molecule may contain at least
one nucleomonomer that is a modified nucleotide analogue. The
nucleotide analogues may be located at positions where the
target-specific activity, e.g., the RNAi mediating activity is not
substantially effected, e.g., in a region at the 5'-end or the
3'-end of the double-stranded molecule, where the overhangs may be
stabilized by incorporating modified nucleotide analogues.
In another aspect, double-stranded RNA molecules known in the art
can be used in methods of the present invention. Double-stranded
RNA molecules known in the art may also be modified according to
the teachings herein in conjunction with such methods, e.g., by
using modified nucleomonomers. For example, see U.S. Pat. No.
6,506,559; U.S. 2002/0,173,478 A1; U.S. 2002/0,086,356 A1; Shuey,
et al., "RNAi: gene-silencing in therapeutic intervention." Drug
Discov. Today 2002 Oct. 15; 7(20):1040-6; Aoki, et al., "Clin. Exp.
Pharmacol. Physiol. 2003 January; 30(1-2):96-102; Cioca, et al.,
"RNA interference is a functional pathway with therapeutic
potential in human myeloid leukemia cell lines. Cancer Gene Ther.
2003 February; 10(2): 125-33.
Further examples of double-stranded RNA molecules include those
disclosed in the following references: Kawasaki, et al., "Short
hairpin type of dsRNAs that are controlled by tRNA(Val) promoter
significantly induce RNAi-mediated gene silencing in the cytoplasm
of human cells." Nucleic Acids Res. 2003 Jan. 15; 31(2):700-7;
Cottrell, et al., "Silence of the strands: RNA interference in
eukaryotic pathogens." Trends Microbiol. 2003 January; 11(1):37-43;
Links, "Mammalian RNAi for the masses." Trends Genet. 2003 January;
19(1):9-12; Hamada, et al., "Effects on RNA interference in gene
expression (RNAi) in cultured mammalian cells of mismatches and the
introduction of chemical modifications at the 3'-ends of siRNAs."
Antisense Nucleic Acid Drug Dev. 2002 October; 12(5):301-9; Links,
"RNAi and related mechanisms and their potential use for therapy."
Curr. Opin. Chem. Biol. 2002 December; 6(6):829-34; Kawasaki, et
al., "Short hairpin type of dsRNAs that are controlled by tRNA(Val)
promoter significantly induce RNAi-mediated gene silencing in the
cytoplasm of human cells." Nucleic Acids Res. 2003 Jan. 15;
31(2):700-7).
A nick is two non-linked nucleomonomers in an oligonucleotide. A
nick can be included at any point along the sense or antisense
nucleotide sequence. In a preferred embodiment, a nick is in the
sense sequence. In another preferred embodiment, the nick is at
least about four nucleomonomers in from an end of the duplexed
region of the oligonucleotide (e.g., is at least about four
nucleomonomers away from the 5' or 3' end of the oligonucleotide or
away from a loop structure. For example, in one embodiment, the
nick is present in the middle of the sense strand of the duplex
molecule (e.g., if the sense sequence of the duplex is 30
nucleomonomers in length, nucleomonomers 14 and 15 or 15 and 16 are
unlinked). In an embodiment, a nick may optionally be ligated to
form a circular nucleic acid molecule.
For example, in the structure shown in FIG. 5, the indicated U
nucleomonomer is not bonded to the neighboring nucleomonomer, e.g.,
by a phosphodiester bond. The 5' OH of the nick may optionally be
phosphorylated to allow enzymatic ligation of the oligonucleotide
into a circle.
As used herein, the term "nucleotide" includes any monomeric unit
of DNA or RNA containing a sugar moiety (pentose), a phosphate, and
a nitrogenous heterocyclic base. The base is usually linked to the
sugar moiety via the glycosidic carbon (at the 1' carbon of
pentose) and that combination of base and sugar is called a
"nucleoside." The base characterizes the nucleotide with the four
customary bases of DNA being adenine (A), guanine (G), cytosine (C)
and thymine (T). Inosine (I) is an example of a synthetic base that
can be used to substitute for any of the four, naturally-occurring
bases (A, C, G, or T). The four RNA bases are A, G, C, and uracil
(U). Accordingly, an oligonucleotide may be a nucleotide sequence
comprising a linear array of nucleotides connected by
phosphodiester bonds between the 3' and 5' carbons of adjacent
pentoses. Other modified nucleosides/nucleotides are described
herein and may also be used in the oligonucleotides of the
invention.
Oligonucleotides may comprise, for example, oligonucleotides,
oligonucleosides, polydeoxyribonucleotides (containing
2'-deoxy-D-ribose) or modified forms thereof, e.g., DNA,
polyribonucleotides (containing D-ribose or modified forms
thereof), RNA, or any other type of polynucleotide which is an
N-glycoside or C-glycoside of a purine or pyrimidine base, or
modified purine or pyrimidine base. The term oligonucleotide
includes compositions in which adjacent nucleomonomers are linked
via phosphorothioate, amide or other linkages (e.g., Neilsen, P.
E., et al. 1991. Science. 254:1497). Generally, the term "linkage"
refers to any physical connection, preferably covalent coupling,
between two or more nucleic acid components, e.g., catalyzed by an
enzyme such as a ligase.
In addition to its art-recognized meaning (e.g., a relatively short
length single or double-stranded sequences of deoxyribonucleotides
or ribonucleotides linked via phosphodiester bonds), the term
"oligonucleotide" includes any structure that serves as a scaffold
or support for the bases of the oligonucleotide, where the scaffold
permits binding to the target nucleic acid molecule in a
sequence-dependent manner.
Oligonucleotides of the invention are isolated. The term "isolated"
includes nucleic acid molecules which are synthesized (e.g.,
chemically, enzymatically, or recombinantly) or are naturally
occurring but separated from other nucleic acid molecules which are
present in a natural source of the nucleic acid. Preferably, a
naturally occurring "isolated" nucleic acid molecule is free of
sequences which naturally flank the nucleic acid molecule (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid
molecule) in a nucleic acid molecule in an organism from which the
nucleic acid molecule is derived.
The term "nucleomonomer" includes a single base covalently linked
to a second moiety. Nucleomonomers include, for example,
nucleosides and nucleotides. Nucleomonomers can be linked to form
oligonucleotides that bind to target nucleic acid sequences in a
sequence specific manner.
In one embodiment, modified (non-naturally occurring)
nucleomonomers can be used in the oligonucleotides described
herein. For example, nucleomonomers may be based on bases (purines,
pyrimidines, and derivatives and analogs thereof) bound to
substituted and unsubstituted cycloalkyl moieties, e.g., cyclohexyl
or cyclopentyl moieties, and substituted and unsubstituted
heterocyclic moieties, e.g., 6-member morpholino moieties or,
preferably, sugar moieties.
Sugar moieties include natural, unmodified sugars, e.g.,
monosaccharides (such as pentoses, e.g., ribose, deoxyribose),
modified sugars and sugar analogs. Possible modifications of
nucleomonomers, particularly of a sugar moiety, include, for
example, replacement of one or more of the hydroxyl groups with a
halogen, a heteroatom, an aliphatic group, or the functionalization
of the hydroxyl group as an ether, an amine, a thiol, or the like.
One particularly useful group of modified nucleomonomers are
2'-O-methyl nucleotides, especially when the 2'-O-methyl
nucleotides are used as nucleomonomers in the ends of the
oligomers. Such 2'O-methyl nucleotides may be referred to as
"methylated," and the corresponding nucleotides may be made from
unmethylated nucleotides followed by alkylation or directly from
methylated nucleotide reagents. Modified nucleomonomers may be used
in combination with unmodified nucleomonomers. For example, an
oligonucleotide of the invention may contain both methylated and
unmethylated nucleomonomers.
Some exemplary modified nucleomonomers include sugar- or
backbone-modified ribonucleotides. Modified ribonucleotides may
contain a nonnaturally occurring base (instead of a naturally
occurring base) such as uridines or cytidines modified at the
5-position, e.g., 5-(2-amino)propyl uridine and 5-bromo uridine;
adenosines and guanosines modified at the 8-position, e.g., 8-bromo
guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; and
N-alkylated nucleotides, e.g., N6-methyl adenosine. Also,
sugar-modified ribonucleotides may have the 2'-OH group replaced by
a H, alxoxy (or OR), R or alkyl, halogen, SH, SR, amino (such as
NH.sub.2, NHR, NR.sub.2), or CN group, wherein R is lower alkyl,
alkenyl, or alkynyl.
Modified ribonucleotides may also have the phosphoester group
connecting to adjacent ribonucleotides replaced by a modified
group, e.g., of phosphothioate group. More generally, the various
nucleotide modifications may be combined.
In one embodiment, sense oligomers may have 2' modifications on the
ends (1 on each end, 2 on each end, 3 on each end, and 4 on each
end, and so on; as well as 1 on one end, 2 on one end, 3 on one
end, and 4 on one end, and so on; and even unbalanced combinations
such as 1 on one end and 2 on the other end, and so on). Likewise,
the antisense strand may have 2' modifications on the ends (1 on
each end, 2 on each end, 3 on each end, and 4 on each end, and so
on; as well as 1 on one end, 2 on one end, 3 on one end, and 4 on
one end, and so on; and even unbalanced combinations such as 1 on
one end and 2 on the other end, and so on). In preferred aspects,
such 2'-modifications are in the sense RNA strand or the sequences
other than the antisense strand.
To further maximize endo- and exonuclease resistance, in addition
to the use of 2' modified nucleomonomers in the ends,
inter-nucleomonomer linkages other than phosphodiesters may be
used. For example, such end blocks may be used alone or in
conjunction with phosphothioate linkages between the 2'-O-methyl
linkages. Preferred 2'-modified nucleomonomers are 2'-modified C
and U bases.
Although the antisense strand may be substantially identical to at
least a portion of the target gene (or genes), at least with
respect to the base pairing properties, the sequence need not be
perfectly identical to be useful, e.g., to inhibit expression of a
target gene's phenotype. Generally, higher homology can be used to
compensate for the use of a shorter antisense gene. In some cases,
the antisense strand generally will be substantially identical
(although in antisense orientation) to the target gene.
One particular example of a composition of the invention has
end-blocks on both ends of a sense oligonucleotide and only the 3'
end of an antisense oligonucleotide. Without wishing to be bound by
theory, the inventors believe that a 2'-O-modified sense strand
works less well than unmodified because it is not efficiently
unwound. Accordingly, another embodiment of the invention includes
duplexes in which nucleomonomer-nucleomonomer mismatches are
present in a sense 2'-O-methyl strand (and are thought to be easier
to unwind).
Accordingly, for a given first oligonucleotide strand, a number of
complementary second oligonucleotide strands are permitted
according to the invention. For example, in the following Tables, a
targeted and a non-targeted oligonucleotide are illustrated with
several possible complementary oligonucleotides. The individual
nucleotides may be 2'-OH RNA nucleotides (R) or the corresponding
2'-O-methyl nucleotides (M), and the oligonucleotides themselves
may contain mismatched nucleotides (lower case letters).
Targeted Oligonucleotide:
TABLE-US-00001 (SEQ ID NO: 2) First Strand: CCCUUCUGUCUUGAACAUGAG
(SEQ ID NO: 3) Second Strand: CTgATGTTCAAGACAGAAcGG (methyl groups
.fwdarw.) MMMMMMMMMMMMMMMMMMMMM (SEQ ID NO: 4)
CTgATGTTCAAGACAGAAcGG RRRRRRRRRRRRRRRRRRRDD (SEQ ID NO: 5)
CTCAUGUUCAAGACAGAAGGG RRRRRRMMMMMMMMMRRRRRR (SEQ ID NO: 6)
CTCAUGUUCAAGACAGAAGGG MMMMMMRRRRRRRRRMMMMMM (SEQ ID NO: 7)
CTCAUGUUCAAGACAGAAGGG RMRMRMRMRMRMRMRMRMRMR
Non-Targeted Oligonucleotide:
TABLE-US-00002 (SEQ ID NO: 8) First Strand: GAGTACAAGTTCTGTCTTCCC
(SEQ ID NO: 9) Second Strand: GGcAAGACAGAACTTGTAgTC (methyl groups
.fwdarw.) MMMMMMMMMMMMMMMMMMMMM (SEQ ID NO: 10)
GGGAAGACAGAACTTGTACTC RRRRRRMMMMMMMMMRRRRRR (SEQ ID NO: 11)
GGGAAGACAGAACTTGTACTC MMMMMMRRRRRRRRRMMMMMM (SEQ ID NO: 12)
GGGAAGACAGAACTTGTACTC RMRMRMRMRMRMRMRMRMRMR
Another example of further modifications that may be used in
conjunction with 2'-O-methyl nucleomonomers are modification of the
sugar residues themselves, for example alternating modified and
unmodified sugars, particularly in the sense strand.
The invention further includes double stranded nucleic acid
molecules (e.g., RNA molecules) which have structures defined by
the following formula:
TABLE-US-00003 First Strand X.sub.15-30 Second Strand
A.sub.0-25X.sub.0-25B.sub.0-25
In the formula set out above, X, A, and B are nucleotides (e.g., A,
G, C, U, etc.). Also, either of the first strand or the second
strand may be a sense strand. As a results, either of the first
strand or the second strand may be an antisense strand. Further, X
is typically a nucleotide which has no modifications on the base or
sugar. Further, A and/or B are nucleotides which may independently
contain one or more base or sugar modifications. These
modifications may be any modifications known in the art or
described elsewhere herein. Examples of sugar modifications include
ribose modifications at the 2' position such as 2'-O-propyl (P),
2'-O-methyl (M), 2'-O-ethyl (E), and 2'-fluoro (F). Generic
examples of nucleic acid molecules of the invention include those
with the following:
TABLE-US-00004 XXXXXXXXXXXXXXX XXXXX AXXXXXXXXXXXXXX XXXXB
XXXXXXXXXXXXXXX XXXXX AAXXXXXXXXXXXXX XXXBB XXXXXXXXXXXXXXX XXXXX
AAAXXXXXXXXXXXX XXBBB XXXXXXXXXXXXXXX XXXXX AAAAXXXXXXXXXXX XBBBB
XXXXXXXXXXXXXXX XXXXX AAAAXXXXXXXXXXX XXXBB XXXXXXXXXXXXXXX XXXXX
AAXXXXXXXXXXXXX BBBBB XXXXXXXXXXXXXXX XXXXX AAAAAAAAAAAAAAA AAAAA
XXXXXXXXXXXXXXX XXXXX AAAAAAAXXXBBBBBB BBBB
Examples of nucleic acid molecules of the invention which contain
specific modifications include those with the following
modifications, in which X represents an unmodified nucleotide, P
represents 2'-O-propyl, M represents 2'-O-methyl, E represents
2'-O-ethyl, and F represents 2'-fluoro:
TABLE-US-00005 XXXXXXXXXXXXXXXXXXXXXXX XX PPMMXXXXXXXXXXXXXXXXEEM
MM XXXXXXXXXXXXXXXXXXXXXXX XX EEEEXXXXXXXXXXXXXXXXEEM MM
XXXXXXXXXXXXXXXXXXXXXXX XX PPEEXXXXXXXXXXXXXXXXEEM MM
XXXXXXXXXXXXXXXXXXXXXXX XX EEEEEXXXXXXXXXXXXXXXEEEE E
XXXXXXXXXXXXXXXXXXXXXXX XX PPPPPPPXXXXXXXXXXXPPPPPPP
XXXXXXXXXXXXXXXXXXXXXXX XX FFPPPXXXXXXXXXXXXXXXPPPFF
XXXXXXXXXXXXXXXXXXXXXXX XX MPPPPPPPPPPPPPPPPXXXPPPPM
XXXXXXXXXXXXXXXXXXXXXXX XX FFFFFXXXXXXXXXXXXXXXFFFFF
XXXXXXXXXXXXXXXXXXXXXXX XX PEEPEEMPXXXXXXXXXPMEEPEEP
XXXXXXXXXXXXXXXXXXXXXXX XX MEXXXXXXXXXXXXXXMMMMM MMMM
XXXXXXXXXXXXXXXXXXXXXXX XX MXXXXXXXXXXXXXXXMMMMM MMMM
XXXXXXXXXXXXXXXXXXXXXXX XX EEXXXXXXXXXXXXXXXEEEEEEE E
In some embodiments, the length of the sense strand can be 29, 28,
27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides. Similarly,
the length of the antisense strand can be 29, 28, 27, 26, 25, 24,
23, 22, 21, 20, 19, or 18 nucleotides. Further, when a
double-stranded nucleic acid molecule is formed from such sense and
antisense molecules, the resulting duplex may have blunt ends or
overhangs of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14
nucleotides on one end or independently on each end. Further,
double stranded nucleic acid molecules of the invention may be
composed of a sense strand and an antisense strand wherein these
strands are of lengths described above, and are of the same or
different lengths, but share only 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 nucleotides of sequence complementarity. By way of
illustration, in a situation where the sense strand is 20
nucleotides in length and the antisense is 25 nucleotides in length
and the two strands share only 15 nucleotides of sequence
complementarity, a double stranded nucleic acid molecules may be
formed with a 10 nucleotide overhang on one end and a 5 nucleotide
overhang on the other end.
Double-stranded oligonucleotides of the invention include
STEALTH.TM. RNAs which may be obtained from either Sequitur Inc.
(Natick, Mass.), recently acquired by Invitrogen Corporation
(Carlsbad, Calif.) or Invitrogen Corporation directly. STEALTH.TM.
RNAs are often synthesized based upon nucleotide sequence
information provided by purchasers. In particular instances,
purchasers may provide the nucleotide sequence of an RNA transcript
for which "knockdown" is desired and Invitrogen Corporation then
selects suitable STEALTH.TM. RNA for the particular application or
purchasers may provide the actual sequence of the STEALTH.TM. RNAs
to be used in the "knockdown" process. Typically, in the second
instance, the nucleotide sequences provided by purchasers are
between 20 and 30 nucleotides in length. A more detailed
description of business method aspects of the invention is set out
elsewhere herein. However, these business methods typically
include, in part, providing STEALTH.TM. RNA, as well as protocols
and additional reagents and compounds for purchasers to use the
purchased STEALTH.TM. RNA for knocking down gene expression.
As a further example, the use of 2'-O-methyl RNA may be used
beneficially in circumstances in which it is desirable to minimize
cellular stress responses. RNA having 2'-O-methyl nucleomonomers
may not be recognized by cellular machinery that is thought to
recognize unmodified RNA. The use of 2'-O-methylated or partially
2'-O-methylated RNA may avoid the interferon response to
double-stranded nucleic acids, while maintaining target RNA
inhibition. This RNAi ("stealth RNA") is useful, for example, for
avoiding the interferon or other cellular stress responses, both in
short RNAi (e.g., siRNA) sequences that induce the interferon
response, and in longer RNAi sequences that may induce the
interferon response.
An especially advantageous use of the present invention is in gene
function studies in which multiple RNAi sequences are used.
According to present methods known in the art, frequently there is
no way of predicting which nucleic acid sequences might induce a
stress response, including the interferon response, and in this
regard the present invention significantly advances the state of
the art. For example, if all of the multiple sequences are
partially 2'-O-methylated, the stress response, including
interferon response, may be avoided, and thus avoid confounding
results in which some sequences affect cellular phenotype
independent of the target gene inhibition. Other chemical
modifications in addition to 2'-O-methylation may also achieve this
effect.
For example, modified sugars include D-ribose, 2'-O-alkyl
(including 2'-O-methyl and 2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino,
2'-S-alkyl, 2'-halo (including 2'-fluoro), 2'-methoxyethoxy,
2'-allyloxy (--OCH.sub.2CH.dbd.CH.sub.2), 2'-propargyl, 2'-propyl,
ethynyl, ethenyl, propenyl, and cyano and the like. In one
embodiment, the sugar moiety can be a hexose and incorporated into
an oligonucleotide as described (Augustyns, K., et al., Nucl.
Acids. Res. 18:4711 (1992)). Exemplary nucleomonomers can be found,
e.g., in U.S. Pat. No. 5,849,902, incorporated by reference
herein.
The term "alkyl" includes saturated aliphatic groups, including
straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl,
pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain
alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl
(alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl,
cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and
cycloalkyl substituted alkyl groups. In certain embodiments, a
straight chain or branched chain alkyl has 6 or fewer carbon atoms
in its backbone (e.g., C.sub.1-C.sub.6 for straight chain,
C.sub.3-C.sub.6 for branched chain), and more preferably 4 or
fewer. Likewise, preferred cycloalkyls have from 3-8 carbon atoms
in their ring structure, and more preferably have 5 or 6 carbons in
the ring structure. The term C.sub.1-C.sub.6 includes alkyl groups
containing 1 to 6 carbon atoms.
Moreover, unless otherwise specified, the term alkyl includes both
"unsubstituted alkyls" and "substituted alkyls," the latter of
which refers to alkyl moieties having independently selected
substituents replacing a hydrogen on one or more carbons of the
hydrocarbon backbone. Such substituents can include, for example,
alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety. Cycloalkyls can be further
substituted, e.g., with the substituents described above. An
"alkylaryl" or an "arylalkyl" moiety is an alkyl substituted with
an aryl (e.g., phenylmethyl (benzyl)). The term "alkyl" also
includes the side chains of natural and unnatural amino acids. The
term "n-alkyl" means a straight chain (i.e., unbranched)
unsubstituted alkyl group.
The term "alkenyl" includes unsaturated aliphatic groups analogous
in length and possible substitution to the alkyls described above,
but that contain at least one double bond. For example, the term
"alkenyl" includes straight-chain alkenyl groups (e.g., ethylenyl,
propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl,
decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl
(alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl,
cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted
cycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted
alkenyl groups. In certain embodiments, a straight chain or
branched chain alkenyl group has 6 or fewer carbon atoms in its
backbone (e.g., C.sub.2-C.sub.6 for straight chain, C.sub.3-C.sub.6
for branched chain). Likewise, cycloalkenyl groups may have from
3-8 carbon atoms in their ring structure, and more preferably have
5 or 6 carbons in the ring structure. The term C.sub.2-C.sub.6
includes alkenyl groups containing 2 to 6 carbon atoms.
Moreover, unless otherwise specified, the term alkenyl includes
both "unsubstituted alkenyls" and "substituted alkenyls," the
latter of which refers to alkenyl moieties having independently
selected substituents replacing a hydrogen on one or more carbons
of the hydrocarbon backbone. Such substituents can include, for
example, alkyl groups, alkynyl groups, halogens, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
The term "alkynyl" includes unsaturated aliphatic groups analogous
in length and possible substitution to the alkyls described above,
but which contain at least one triple bond. For example, the term
"alkynyl" includes straight-chain alkynyl groups (e.g., ethynyl,
propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl,
decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or
cycloalkenyl substituted alkynyl groups. In certain embodiments, a
straight chain or branched chain alkynyl group has 6 or fewer
carbon atoms in its backbone (e.g., C.sub.2-C.sub.6 for straight
chain, C.sub.3-C.sub.6 for branched chain). The term
C.sub.2-C.sub.6 includes alkynyl groups containing 2 to 6 carbon
atoms.
Moreover, unless otherwise specified, the term alkynyl includes
both "unsubstituted alkynyls" and "substituted alkynyls," the
latter of which refers to alkynyl moieties having independently
selected substituents replacing a hydrogen on one or more carbons
of the hydrocarbon backbone. Such substituents can include, for
example, alkyl groups, alkynyl groups, halogens, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
Unless the number of carbons is otherwise specified, "lower alkyl"
as used herein means an alkyl group, as defined above, but having
from one to five carbon atoms in its backbone structure. "Lower
alkenyl" and "lower alkynyl" have chain lengths of, for example,
2-5 carbon atoms.
The term "alkoxy" includes substituted and unsubstituted alkyl,
alkenyl, and alkynyl groups covalently linked to an oxygen atom.
Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy,
propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxy
groups include halogenated alkoxy groups. The alkoxy groups can be
substituted with independently selected groups such as alkenyl,
alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.
Examples of halogen substituted alkoxy groups include, but are not
limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,
chloromethoxy, dichloromethoxy, trichloromethoxy, etc.
The term "heteroatom" includes atoms of any element other than
carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,
sulfur and phosphorus.
The term "hydroxy" or "hydroxyl" includes groups with an --OH or
--O.sup.- (with an appropriate counterion).
The term "halogen" includes fluorine, bromine, chlorine, iodine,
etc. The term "perhalogenated" generally refers to a moiety wherein
all hydrogens are replaced by halogen atoms.
The term "substituted" includes independently selected substituents
which can be placed on the moiety and which allow the molecule to
perform its intended function. Examples of substituents include
alkyl, alkenyl, alkynyl, aryl, (CR'R'').sub.0-3NR'R'',
(CR'R'').sub.0-3CN, NO.sub.2, halogen,
(CR'R'').sub.0-3C(halogen).sub.3,
(CR'R'').sub.0-3CH(halogen).sub.2,
(CR'R'').sub.0-3CH.sub.2(halogen), (CR'R'').sub.0-3CONR'R'',
(CR'R'').sub.0-3S(O).sub.1-2NR' R'', (CR'R'').sub.0-3CHO,
(CR'R'').sub.0-3O(CR'R'').sub.0-3H, (CR'R'').sub.0-3
S(O).sub.0-2R', (CR'R'').sub.0-3O(CR'R'').sub.0-3H,
(CR'R'').sub.0-3COR', (CR'R'').sub.0-3CO.sub.2R', or
(CR'R'').sub.0-30R' groups; wherein each R' and R'' are each
independently hydrogen, a C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5
alkenyl, C.sub.2-C.sub.5 alkynyl, or aryl group, or R' and R''
taken together are a benzylidene group or a
--(CH.sub.2).sub.2O(CH.sub.2).sub.2-- group.
The term "amine" or "amino" includes compounds or moieties in which
a nitrogen atom is covalently bonded to at least one carbon or
heteroatom. The term "alkyl amino" includes groups and compounds
wherein the nitrogen is bound to at least one additional alkyl
group. The term "dialkyl amino" includes groups wherein the
nitrogen atom is bound to at least two additional alkyl groups.
The term "ether" includes compounds or moieties which contain an
oxygen bonded to two different carbon atoms or heteroatoms. For
example, the term includes "alkoxyalkyl," which refers to an alkyl,
alkenyl, or alkynyl group covalently bonded to an oxygen atom which
is covalently bonded to another alkyl group.
The term "base" includes the known purine and pyrimidine
heterocyclic bases, deazapurines, and analogs (including
heterocyclic substituted analogs, e.g., aminoethyoxy phenoxazine),
derivatives (e.g., 1-alkyl-, 1-alkenyl-, heteroaromatic- and
1-alkynyl derivatives) and tautomers thereof. Examples of purines
include adenine, guanine, inosine, diaminopurine, and xanthine and
analogs (e.g., 8-oxo-N.sup.6-methyladenine or 7-diazaxanthine) and
derivatives thereof. Pyrimidines include, for example, thymine,
uracil, and cytosine, and their analogs (e.g., 5-methylcytosine,
5-methyluracil 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine and
4,4-ethanocytosine). Other examples of suitable bases include
non-purinyl and non-pyrimidinyl bases such as 2-aminopyridine and
triazines.
In a preferred embodiment, the nucleomonomers of an oligonucleotide
of the invention are RNA nucleotides. In another preferred
embodiment, the nucleomonomers of an oligonucleotide of the
invention are modified RNA nucleotides. Thus, the oligonucleotides
contain modified RNA nucleotides.
The term "nucleoside" includes bases which are covalently attached
to a sugar moiety, preferably ribose or deoxyribose. Examples of
preferred nucleosides include ribonucleosides and
deoxyribonucleosides. Nucleosides also include bases linked to
amino acids or amino acid analogs which may comprise free carboxyl
groups, free amino groups, or protecting groups. Suitable
protecting groups are well known in the art (see P. G. M. Wuts and
T. W. Greene, "Protective Groups in Organic Synthesis", 2.sup.nd
Ed., Wiley-Interscience, New York, 1999).
The term "nucleotide" includes nucleosides which further comprise a
phosphate group or a phosphate analog.
As used herein, the term "linkage" includes a naturally occurring,
unmodified phosphodiester moiety (--O--(PO.sub.2.sup.-)--O--) that
covalently couples adjacent nucleomonomers. As used herein, the
term "substitute linkage" includes any analog or derivative of the
native phosphodiester group that covalently couples adjacent
nucleomonomers. Substitute linkages include phosphodiester analogs,
e.g., phosphorothioate, phosphorodithioate, and
P-ethyoxyphosphodiester, P-ethoxyphosphodiester,
P-alkyloxyphosphotriester, methylphosphonate, and nonphosphorus
containing linkages, e.g., acetals and amides. Such substitute
linkages are known in the art (e.g., Bjergarde et al. 1991. Nucleic
Acids Res. 19:5843; Caruthers et al. 1991. Nucleosides Nucleotides.
10:47).
In certain embodiments, oligonucleotides of the invention comprise
3' and 5' termini (except for circular oligonucleotides). In one
embodiment, the 3' and 5' termini of an oligonucleotide can be
substantially protected from nucleases e.g., by modifying the 3' or
5' linkages (e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). For
example, oligonucleotides can be made resistant by the inclusion of
a "blocking group." The term "blocking group" as used herein refers
to substituents (e.g., other than OH groups) that can be attached
to oligonucleotides or nucleomonomers, either as protecting groups
or coupling groups for synthesis (e.g., FITC, propyl
(CH.sub.2--CH.sub.2--CH.sub.3), glycol (--O--CH2-CH2-O--)phosphate
(PO.sub.3.sup.2), hydrogen phosphonate, or phosphoramidite).
"Blocking groups" also include "end blocking groups" or
"exonuclease blocking groups" which protect the 5' and 3' termini
of the oligonucleotide, including modified nucleotides and
non-nucleotide exonuclease resistant structures.
Exemplary end-blocking groups include cap structures (e.g., a
7-methylguanosine cap), inverted nucleomonomers, e.g., with 3'-3'
or 5'-5' end inversions (see, e.g., Ortiagao et al. 1992. Antisense
Res. Dev. 2:129), methylphosphonate, phosphoramidite,
non-nucleotide groups (e.g., non-nucleotide linkers, amino linkers,
conjugates) and the like. The 3' terminal nucleomonomer can
comprise a modified sugar moiety. The 3' terminal nucleomonomer
comprises a 3'-O that can optionally be substituted by a blocking
group that prevents 3'-exonuclease degradation of the
oligonucleotide. For example, the 3'-hydroxyl can be esterified to
a nucleotide through a 3'.fwdarw.3' internucleotide linkage. For
example, the alkyloxy radical can be methoxy, ethoxy, or
isopropoxy, and preferably, ethoxy. Optionally, the 3'.fwdarw.3'
linked nucleotide at the 3' terminus can be linked by a substitute
linkage. To reduce nuclease degradation, the 5' most 3'.fwdarw.5'
linkage can be a modified linkage, e.g., a phosphorothioate or a
P-alkyloxyphosphotriester linkage. Preferably, the two 5' most
3'.fwdarw.5' linkages are modified linkages. Optionally, the 5'
terminal hydroxy moiety can be esterified with a phosphorus
containing moiety, e.g., phosphate, phosphorothioate, or
P-ethoxyphosphate.
In one embodiment, the sense strand of an oligonucleotide comprises
a 5' group that allows for RNAi activity but which renders the
sense strand inactive in terms of gene targeting. Preferably, such
a 5' modifying group is a phosphate group or a group larger than a
phosphate group. Oligonucleotides of this type often exhibit
increased specificity for a target gene in a cell that corresponds
to the nucleotide sequence of the antisense strand. This is because
the sense strand in such an oligonucleotide is often rendered
incapable of mediating cleavage of any nucleotide sequence it might
bind to non-specifically and thus will not inactivate any other
genes in the cell. Thus, observed decrease in the expression of a
gene within a cell transfected with such an oligonucleotide will
often be attributed to the direct or indirect effect of the
anti-sense strand. The term "specificity for a target gene," as
used herein means the extent to which an effect of an
oligonucleotide on a cell can be attributed directly or indirectly
to the inhibition of expression of a target gene by an antisense
nucleotide sequence present in said oligonucleotide.
Thus, according to another embodiment, the invention provides a
method of increasing the specificity of an oligonucleotide for a
target gene in a cell, wherein said oligonucleotide comprises a
sense strand and an antisense strand, wherein both the sense strand
and the antisense strand are capable of binding to corresponding
nucleotide sequences if present in said cell, said method
comprising the step of modifying the 5' terminal hydroxy moiety of
said sense strand with a phosphate group or a group larger than a
phosphate group prior to contacting said oligonucleotide with said
cell so as to render said sense strand incapable of mediating
cleavage of any nucleotide sequence it might bind to
non-specifically and thus will not inactivate any other genes in
the cell.
The invention also provides an improvement in a method of
regulating the expression of a target gene in a cell, comprising
contacting a cell with an oligonucleotide comprising a sense strand
and an antisense strand, wherein both the sense strand and the
antisense strand are capable of binding to corresponding nucleotide
sequences if present in said cell, said improvement comprising the
step of modifying the 5' terminal hydroxy moiety of said sense
strand with a phosphate group or a group larger than a phosphate
group prior to contacting said oligonucleotide with said cell so as
to render said sense strand incapable of binding to corresponding
nucleotide sequences if present in said cell.
In another embodiment, the antisense strand of an oligonucleotide
comprises a 5' phosphate group or a group larger than a phosphate
group. Oligonucleotides in accordance with this aspect of the
invention which comprise such a modification of the antisense
strand typically cannot inactivate a target gene that corresponds
to the nucleotide sequence of said antisense strand. However, such
modified oligonucleotides are extremely useful as controls for
non-specific effects caused by a corresponding oligonucleotide that
lacks such a 5' modification on the antisense strand. Non-specific
effects include all effects on the cell by an oligonucleotide
except those caused directly or indirectly by the inactivation of a
target gene by a corresponding nucleotide sequence present in the
antisense strand of said oligonucleotide.
Thus, according to a related embodiment, the invention provides a
method of determining the non-specific effects of a test
oligonucleotide transfected into a population of cells, wherein
said test oligonucleotide comprises a sense strand, an antisense
strand and at least one modified oligonucleotide, said method
comprising the steps of: a) providing an oligonucleotide having the
same nucleotide sequence and modifications as said test
oligonucleotide and additionally comprising a 5' modification on
the antisense strand, wherein said 5' modification is a phosphate
group or a group larger than a phosphate group; b) transfecting
said population of cells with the oligonucleotide provided in step
a); and c) determining the effect of the oligonucleotide provided
in step a) on said population of cells.
In particular embodiments, the invention comprises methods for
measuring uptake of nucleic acid molecules (e.g., double-stranded
nucleic acid molecules) by cells (e.g., eukaryotic cells). In
particular embodiments, such methods comprise: (a) contacting a
cell or population of cells (e.g., eukaryotic cells) with a
detectably labeled nucleic acid molecule or a population of
detectably labeled nucleic acid molecules (e.g., a mixture of
detectably labeled nucleic acid molecules such as double-stranded
nucleic acid molecules which differ in nucleotide sequence) under
conditions which allow for some or all of the nucleic acid
molecules to enter then cell or cells; (b) quantifying the amount
of detectably labeled which has entered either (i) the cell or (ii)
some or all of the cells of the population, for example by exposing
the cells to one or more wavelengths of light which excite one or
more label (e.g., one or more fluorescent label); and (c) measuring
one or more signal (e.g., one or more fluorescent or other signal)
generated from the label(s) in the cells. In more specific
embodiments, the double-stranded nucleic acid molecule may contain
one or more bound (e.g., covalently bound) label (e.g., one or more
fluorescent label). In additional specific embodiments, the
double-stranded nucleic acid molecule may be between 18 and 30,
between 20 and 30, or between 22 and 30 nucleosides in length.
Further, the double-stranded nucleic acid molecule may be 25
nucleosides in length.
The detectable label employed may be any suitable label known in
the art and include radiolabels, chemiluminescent, and fluorescent
labels. In many instances, the label will be of a type which does
not substantially alter the uptake of the nucleic acid molecules to
which they are attached by cells.
In particular embodiments, one or more label may be located on one
or both 3' ends and/or on one or both 5' ends of the
double-stranded nucleic acid molecules.
Additionally, the signal(s) generated by the label(s) may be
measured by any number of ways, including visually (e.g., by
microscopy) or fluorescent activated cell sorting (FACS). In any
event, measurement of the signal(s) generated label(s) may be used
to determine (a) the number or percentage of cells which have taken
up the label(s), (b) the amount of one or more label taken up by
individual cells or groups of cells, or (c) both (a) and (b).
Labels used in methods and compositions of the invention vary
considerably but will often be labels which are readily detectable.
Examples of such labels include the fluorescent labels FITC and
6-carboxyfluorescein.
Methods of the invention further include those where cells are
contacted with one or more labels for a particular period of time
(e.g., one hour, two hours, three hours, four hours, five hours,
six hours, seven hours, eight hours, nine hours, ten hours, from
about one hour to about ten hours, from about three hours to about
eight hours, from about four hours to about seven hours, etc.) and
then the cells are examined for the presence of the label.
The invention additionally includes methods for determining the
ratio of viable to non-viable cells (e.g., eukaryotic cells) in
populations of cells. In particular aspects, such methods include
those which comprise (a) contacting cells of a population with a
double-stranded nucleic acid molecule, (b) contacting the cells of
the population of (a) with a dye which preferentially stains
non-viable cells, and (c) comparing the number of stained cells to
the number of unstained cells to arrive at the ratio of viable to
non-viable cells in the population. In specific embodiments, step
(b) above may performed, ten hours, twelve hours, twenty hours,
twenty-four hours, thirty hours, forty hours, forty-eight hours,
from about ten hours to about forty-eight hours, from about twelve
hours to about twenty-four hours, or from about sixteen hours to
about thirty hours after step (a).
According to another related embodiment, the invention provides a
kit for determining the non-specific effect on a cell of a test
oligonucleotide transfected into said cell, wherein said test
oligonucleotide comprises a sense strand, an antisense strand and
at least one modified oligonucleotide, said kit comprising a first
vessel containing an oligonucleotide having the same nucleotide
sequence and modifications as said test oligonucleotide and
additionally comprising a 5' modification on said antisense strand,
wherein said 5' modification is a phosphate group or a group larger
than a phosphate group; and instructions for using said 5'-modified
oligonucleotide to determine the non-specific effect on said cell
of said test oligonucleotide transfected into said cell. In a
preferred embodiment, the kit additionally comprises one or more of
the following: an independent vessel containing a dye which
distinguishes live cells from dead cells in said cell population;
an independent vessel comprising an oligonucleotide known to
inhibit a gene expressed in said cell population; an independent
vessel containing said test oligonucleotide; and an independent
vessel comprising an oligonucleotide having the same nucleotide
sequence and modifications as said test oligonucleotide and
additionally comprising a 5' detectable end blocking group on the
sense strand.
In yet another embodiment, the invention provides an
oligonucleotide of the invention that comprises at least one
modified RNA nucleotide and a detectable moiety on one or both of
the sense strand and the antisense strand. The term "detectable
moiety", as used herein, refers to a chemical moiety that renders
the oligonucleotide detectable (e.g., visibly detectable) within a
cell. In many instances, the detectable moiety is a fluorescent
molecule. In some instances, the detectable moiety is a fluorescent
or chemiluminescent fluorophore. In particular instances, the
detectable moiety is FITC. In another instance, the detectable
moiety is a 5'- or 3'-end blocking group located on one or both of
the sense and the antisense strand. In one instance, at least one
RNA nucleotide in this oligonucleotide contains at least one
chemical modification. These modified RNA nucleotides may contain
one or more 2'-fluoro, 2'-O-methyl, 2'-O-ethyl, and/or 2'-O-propyl
groups. In particular instances, (1) all of the nucleotides of the
antisense strand and/or the sense strand contain 2'-fluoro,
2'-O-methyl, 2'-O-ethyl, and/or 2'-O-propyl groups and (2) the
antisense and/or the sense strand contains FITC as a 3'- and/or
5'-end blocking group. In particular embodiments, the entire sense
strand is 2'-fluoroated, 2'-O-methylated, 2'-O-ethylated, or
2'-O-propylated and both the sense and antisense strands contain
FITC as a 3'- or 5'-end blocking group.
Oligonucleotides of the invention that comprise a detectable moiety
can serve as a control for the uptake of corresponding
oligonucleotides having the same nucleotide sequence (with or
without some or all of the modifications on the nucleotides present
in the blocked oligonucleotide), but lacking the detectable moiety.
Oligonucleotides comprising a detectable moiety are particularly
useful in establishing optimal transfection conditions for cells
that will be treated with the corresponding unblocked or
undetectable oligonucleotide. When a detectable moiety present on
the antisense strand which corresponds to a target gene is a 5'-end
blocking group, that antisense strand is incapable of inhibiting
the expression of that target gene. If the detectable moiety is
present elsewhere on the antisense strand and/or anywhere on the
sense strand, the oligonucleotide may still be capable of
inhibiting expression of the target gene, as well as being
detected. An oligonucleotide capable of inhibiting expression of a
target gene is referred to as an "active oligonucleotide." The
invention includes active oligonucleotides and compositions
comprising such active oligonucleotides.
Some detectable moiety-containing oligonucleotides of the invention
can be detected in a cell nucleus for at least 72 hours following
transfection. Moreover, oligonucleotides comprising a detectable
moiety, when used in parallel with a corresponding active
oligonucleotide (or, alternatively, if the oligonucleotide
comprising a detectable moiety is itself active), provide the best
control for any variation in transfection conditions and reagents
on the day that transfection is performed. Also, the persistence of
these detectable oligonucleotides in the cell is typically highly
correlated with siRNA activity of a corresponding active
oligonucleotide.
Thus, according to a related embodiment, the invention provides a
method of determining the uptake of a test oligonucleotide by a
population of cells using a transfection protocol, wherein the test
oligonucleotide comprises a sense strand, an antisense strand and
at least one modified oligonucleotide, the method comprising the
steps of: a) providing an oligonucleotide having (i) the same
nucleotide sequence as the test oligonucleotide, (ii) the same
number of and/or type of modifications, a different number of
and/or type of modifications, or no modifications, and (iii) a
detectable moiety on one or both of the sense and antisense
strands; b) using the transfection protocol to transfect said
population of cells with the oligonucleotide provided in step a);
and c) using detecting means to determine the number of cells in
said population transfected with oligonucleotide provided in step
a). In one embodiment, the test oligonucleotide additionally
comprises a detectable moiety, is an active oligonucleotide and is
the oligonucleotide provided in step a).
The term "detecting means," as used herein, encompasses any method
of detection that would allow one to quantitatively or
qualitatively the presence of the oligonucleotide comprising a
detectable moiety within a cell. For example, if the detectable
moiety is a fluorescent label, then detecting means would comprise
the use of light source having a wavelength to cause excitation of
said fluorescent label and either a fluorescent microscope fitted
with a filter appropriate to observe the emission from said excited
fluorescent label or a fluorescence activated cell sorter.
According to another related embodiment, the invention provides a
kit for optimizing the uptake of a test oligonucleotide by a
population of cells, wherein said test oligonucleotide comprises a
sense strand, an antisense strand and at least one modified
oligonucleotide, said kit comprising a first vessel containing an
oligonucleotide having the same nucleotide sequence and
modifications as said test oligonucleotide and additionally
comprising a detectable moiety on one or both of said sense and
antisense strands and instructions for using said detectable
moiety-containing oligonucleotide to determine uptake of said test
oligonucleotide by said population of cells. In a preferred
embodiment, the kit additionally comprises one or more of the
following: an independent vessel containing a dye which
distinguishes live cells from dead cells in said cell population;
an independent vessel comprising an oligonucleotide known to
inhibit a gene expressed in said cell population; an independent
vessel containing said test oligonucleotide; and an independent
vessel containing an oligonucleotide having the same nucleotide
sequence and modifications as said test oligonucleotide and
additionally comprising a 5' modification on the antisense strand,
wherein said 5' modification is a phosphate group or a group larger
than a phosphate group, and said 5' modification inactivates said
oligonucleotide.
In each of the kit embodiments, the detectable moiety is preferably
FITC. In kit embodiments that include an independent vessel
containing a dye that distinguishes live cells from dead cells in
said cell population, the dye is preferably dead red stain
(Molecular Probes, Eugene Oreg., ethidium homodimer cat. no.
E-1169).
In one embodiment, the oligonucleotides included in the composition
are high affinity oligonucleotides. The term "high affinity" as
used herein includes oligonucleotides that have a Tm (melting
temperature) of or greater than about 60.degree. C., greater than
about 65.degree. C., greater than about 70.degree. C., greater than
about 75.degree. C., greater than about 80.degree. C. or greater
than about 85.degree. C. The Tm is the midpoint of the temperature
range over which the oligonucleotide separates from the target
nucleotide sequence. At this temperature, 50% helical (hybridized)
versus coil (unhybridized) forms are present. Tm is measured by
using the UV spectrum to determine the formation and breakdown
(melting) of hybridization. Base stacking occurs during
hybridization, which leads to a reduction in UV absorption. Tm
depends both on GC content of the two nucleic acid molecules and on
the degree of sequence complementarity. Tm can be determined using
techniques that are known in the art (see for example, Monia et al.
1993. J. Biol. Chem. 268:145; Chiang et al., 1991. J. Biol. Chem.
266:18162; Gagnor et al. 1987. Nucleic Acids Res. 15:10419; Monia
et al. 1996. Proc. Natl. Acad. Sci. 93:15481; Publisis and Tinoco.
1989. Methods in Enzymology 180:304; Thuong et al. 1987. Proc.
Natl. Acad. Sci. USA 84:5129).
In one embodiment, an oligonucleotide can include an agent which
increases the affinity of the oligonucleotide for its target
sequence. The term "affinity enhancing agent" includes agents that
increase the affinity of an oligonucleotide for its target. Such
agents include, e.g., intercalating agents and high affinity
nucleomonomers. Intercalating agents interact strongly and
nonspecifically with nucleic acids. Intercalating agents serve to
stabilize RNA-DNA duplexes and thus increase the affinity of the
oligonucleotides for their targets. Intercalating agents are most
commonly linked to the 3' or 5' end of oligonucleotides. Examples
of intercalating agents include acridine, chlorambucil,
benzopyridoquinoxaline, benzopyridoindole, benzophenanthridine, and
phenazinium. The agents may also impart other characteristics to
the oligonucleotide, for example, increasing resistance to
endonucleases and exonucleases.
In one embodiment, a high affinity nucleomonomer is incorporated
into an oligonucleotide. The language "high affinity nucleomonomer"
as used herein includes modified bases or base analogs that bind to
a complementary base in a target nucleic acid molecule with higher
affinity than an unmodified base, for example, by having more
energetically favorable interactions with the complementary base,
e.g., by forming more hydrogen bonds with the complementary base.
For example, high affinity nucleomonomer analogs such as
aminoethyoxy phenoxazine (also referred to as a G clamp), which
forms four hydrogen bonds with guanine are included in the term
"high affinity nucleomonomer." A high affinity nucleomonomer is
illustrated in FIG. 6 (see, e.g., Flanagan, et al., 1999. Proc.
Natl. Acad. Sci. 96:3513).
Other exemplary high affinity nucleomonomers are known in the art
and include 7-alkenyl, 7-alkynyl, 7-heteroaromatic-, or
7-alkynyl-heteroaromatic-substituted bases or the like which can be
substituted for adenosine or guanosine in oligonucleotides (see,
e.g., U.S. Pat. No. 5,594,121). Also, 7-substituted deazapurines
have been found to impart enhanced binding properties to
oligonucleotides, i.e., by allowing them to bind with higher
affinity to complementary target nucleic acid molecules as compared
to unmodified oligonucleotides. High affinity nucleomonomers can be
incorporated into the oligonucleotides of the instant invention
using standard techniques.
In another embodiment, an agent that increases the affinity of an
oligonucleotide for its target comprises an intercalating agent. As
used herein, the language "intercalating agent" includes agents
which can bind to a DNA double helix. When covalently attached to
an oligonucleotide of the invention, an intercalating agent
enhances the binding of the oligonucleotide to its complementary
genomic DNA target sequence. The intercalating agent may also
increase resistance to endonucleases and exonucleases.
Exemplary intercalating agents are taught by Helene and Thuong
(1989. Genome 31:413), and include e.g., acridine derivatives
(Lacoste et al. 1997. Nucleic Acids Research. 25:1991; Kukreti et
al. 1997. Nucleic Acids Research. 25:4264); quinoline derivatives
(Wilson et al. 1993. Biochemistry 32:10614); and
benzo[f]quino[3,4-b]quioxaline derivatives (Marchand et al. 1996.
Biochemistry. 35:5022; Escude et al. 1998. Proc. Natl. Acad. Sci.
95:3591).
Intercalating agents can be incorporated into an oligonucleotide
using any convenient linkage. For example, acridine or psoralen can
be linked to the oligonucleotide through any available --OH or --SH
group, e.g., at the terminal 5' position of the oligonucleotide,
the 2' positions of sugar moieties, or an OH, NH.sub.2, COOH, or SH
incorporated into the 5-position of pyrimidines using standard
methods.
In one embodiment, when included in an RNase H activating antisense
nucleotide sequence, an agent that increases the affinity of an
oligonucleotide for its target is not positioned adjacent to an
RNase activating region of the oligonucleotide, e.g., is positioned
adjacent to a non-RNase activating region. Preferably, the agent
that increases the affinity of an oligonucleotide for its target is
placed at a distance as far as possible from the RNase activating
domain of the chimeric antisense sequence such that the specificity
of the chimeric antisense sequence is not altered when compared
with the specificity of a chimeric antisense sequence which lacks
the intercalating compound. In one embodiment, this can be
accomplished by positioning the agent adjacent to a non-RNase
activating region. The specificity of the oligonucleotide can be
tested by demonstrating that transcription of a non-target
sequence, preferably a non-target sequence which is structurally
similar to the target (e.g., has some sequence homology or identity
with the target sequence but which is not identical in sequence to
the target), is not inhibited to a greater degree by an
oligonucleotide comprising an affinity enhancing agent than by an
oligonucleotide directed against the same target that does not
comprise an affinity enhancing agent.
Double-stranded oligonucleotides of the invention may be formed by
a single, self-complementary nucleic acid strand or two separate
complementary nucleic acid strands. Duplex formation can occur
either inside or outside the cell containing the target gene.
As used herein, the term "double-stranded" includes one or more
nucleic acid molecules comprising a region of the molecule in which
at least a portion of the nucleomonomers are complementary and
hydrogen bond to form a duplex.
As used herein, the term "duplex" includes the region of the
double-stranded nucleic acid molecule(s) that is (are) hydrogen
bonded to a complementary sequence.
Double-stranded oligonucleotides of the invention may comprise a
nucleotide sequence that is sense to a target gene and a
complementary sequence that is antisense to the target gene. The
sense and antisense nucleotide sequences correspond to the target
gene sequence, e.g., are identical or are sufficiently identical to
effect target gene inhibition (e.g., are about at least about 98%
identical, 96% identical, 94%, 90% identical, 85% identical, or 80%
identical) to the target gene sequence.
When comprised of two separate complementary nucleic acid
molecules, the individual nucleic acid molecules can be of
different lengths.
In one embodiment, a double-stranded oligonucleotide of the
invention is double-stranded over its entire length, i.e., with no
overhanging single-stranded sequence at either end of the molecule,
i.e., is blunt-ended. In another embodiment, a double-stranded
oligonucleotide of the invention is not double-stranded over its
entire length. For instance, when two separate nucleic acid
molecules are used, one of the molecules, e.g., the first molecule
comprising an antisense sequence, can be longer than the second
molecule hybridizing thereto (leaving a portion of the molecule
single-stranded). Likewise, when a single nucleic acid molecule is
used a portion of the molecule at either end can remain
single-stranded.
In one embodiment, a double-stranded oligonucleotide of the
invention is double-stranded over at least about 70% of the length
of the oligonucleotide. In another embodiment, a double-stranded
oligonucleotide of the invention is double-stranded over at least
about 80% of the length of the oligonucleotide. In another
embodiment, a double-stranded oligonucleotide of the invention is
double-stranded over at least about 90%-95% of the length of the
oligonucleotide. In another embodiment, a double-stranded
oligonucleotide of the invention is double-stranded over at least
about 96%-98% of the length of the oligonucleotide.
In one embodiment, the double-stranded duplex constructs of the
invention can be further stabilized against nucleases by forming
loop structures at the 5' or 3' end of the sense or antisense
strand of the construct. For example, the construct can take the
form shown in FIG. 1, where the Ns are nucleomonomers in
complementary oligonucleotide strands (i.e., the top N strand is
complementary to the bottom N strand) of equal length (e.g.,
between about 12 and about 40 nucleotides in length) and X and Y
are each independently selected from a group consisting of nothing
(i.e., the construct is a blunt ended construct with no loops and
no overhang); from about 1 to about 20 nucleotides of 5' overhang;
from about 1 to about 20 nucleotides of 3' overhang; a GAAA loop
(tetra-loop); and a loop consisting from about 4 to about 20
nucleomonomers (where the nucleomonomers are all either G's or
A's).
The sequence of Ns corresponds to the target gene sequence (e.g.,
is homologous or identical to a nucleotide sequence that is sense
or antisense to the target gene sequence), while the nucleotide
sequence of the loop structure does not correspond to the target
gene sequence.
For example, such loops can comprise all G's and A's and be from
about 4 to about 20 nucleotides in length. In one embodiment, such
a loop can be a tetra-loop having a sequence GAAA as depicted in
FIG. 7.
In one embodiment, the number of Ns is about 27.
In embodiments in which loops are at one or both ends of the
construct, the oligonucleotide can be divided by having a "nick"
which is two non-linked nucleomonomers at any point along the sense
or antisense strand, but preferably along the sense strand.
Preferably, the nick is at least four bases from the nearest end of
the duplexed region (to provide enough thermodynamic
stability).
In another embodiment, a construct of the invention can take the
form depicted in FIG. 8, where the Ns are complementary
nucleomonomers in oligonucleotide strands of equal length (e.g.,
between 12-40 nucleomonomers in length); Zs are nucleomonomers in
complementary oligonucleotide strands of between about 2 and about
8 nucleomonomers in length and which comprise a sequence which can
optionally correspond to the target sequence; and where Ms are
nucleomonomers in complementary oligonucleotide strands of between
about 2 and about 8 nucleomonomers in length and which can
optionally correspond to the target sequence.
Preferably, the Zs and Ms are nucleomonomers selected from the
group consisting of C's and G's to make the end of the duplex more
thermodynamically stable. Ends of duplexes can become single
stranded transiently, and since duplex RNA is more stable than
single-stranded RNA, the enhanced stability of the duplex on the
ends will result in higher nuclease stability.
A preferred sequence for Z or M in the antisense strand is from 2-8
nucleomonomers in length or preferably from 3-4 nucleomonomers in
length, e.g., (from 5' to 3') CC, GG, CG, GC, CCC, GGG, CGG, GCC,
GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG,
GCCC, GGCC, or CCGG. The complementary strand would have the
corresponding complementary sequence.
In still another embodiment, a construct of the invention has the
form depicted in FIG. 3, where Ns are nucleomonomers in
complementary oligonucleotide strands (i.e., the top N strand is
complementary to the bottom N strand) of equal length (e.g., from
between about 12 to about 40 nucleomonomers in length) and X is
selected from the group consisting of nothing (i.e., leaving blunt
ends with no loop or overhang); 1-20 nucleotides of 5' overhang;
1-20 nucleotides of 3' overhang; a GAAA loop (tetra-loop); and a
loop consisting of from about 4 to about 20 nucleomonomers (where
the nucleomonomers are all either G's or A's) and where Ms are
nucleomonomers in complementary oligonucleotide strands of between
about 2 and about 8 nucleomonomers in length (which can optionally
correspond to the target sequence). Preferably, Ms are
nucleomonomers selected from the group consisting of contain C's
and G's.
A preferred sequence for M in the antisense strand is from 2-8
nucleomonomers in length or preferably from 3-4 nucleomonomers in
length, e.g., (from 5' to 3') CC, GG, CG, GC, CCC, GGG, CGG, GCC,
GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG,
GCCC, GGCC, or CCGG and the corresponding complement on the
opposite strand.
In another embodiment, the construct can take the form depicted in
FIG. 4, where Ns are nucleomonomers in complementary
oligonucleotide strands of equal length (e.g., from between about
12 to about 40 nucleomonomers in length) and Y is selected from the
group consisting of nothing (i.e., leaving blunt ends with no loop
or overhang; 1-20 nucleotides of 5' overhang; 1-20 nucleotides of
3' overhang; a GAAA loop (tetra-loop); and a loop consisting of a
sequence of from about 4 to about 20 nucleomonomers (where the
nucleomonomers are all either Gs or A's) and where Zs are
nucleomonomers in complementary oligonucleotide strands of between
about 2 and about 8 nucleomonomers in length and which comprise a
sequence which can optionally correspond to the target sequence.
Preferably, the Zs are nucleomonomers selected from the group
consisting of Cs and Gs to make the end of the duplex more
stable.
A preferred sequence for Z in the antisense strand is from 2-8
nucleomonomers in length or preferably from 3-4 nucleomonomers in
length, e.g., (from 5' to 3') CC, GG, CG, GC, CCC, GGG, CGG, GCC,
GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG,
GCCC, GGCC or CCGG (and the corresponding complement on the
opposite strand). For example, in the structure shown in FIG. 9,
GGCC on the end (and its complement) confers additional
stability.
The invention also relates to a double-stranded oligonucleotide
composition having the following structure depicted in FIG. 10,
wherein (1) oligoA is an oligonucleotide of a number of
nucleomonomers; (2) oligoB is an oligonucleotide that has the same
number of nucleomonomers as oligoA and that is complementary to
oligoA; (3) either oligoA or oligoB corresponds to a target gene
sequence.
In this structure, X may be selected from (a) nothing; (b) an
oligonucleotide of about 1 to about 20 nucleotides covalently
bonded to the 5' end of oligoA and constituting a 5' overhang; (c)
an oligonucleotide of about 1 to about 20 nucleotides covalently
bonded to the 3' end of oligoB and constituting a 3' overhang; (d)
and an oligonucleotide of about 4 to about 20 nucleomonomers
covalently bonded to the 3' end of oligoB and the 5' end of oligoA
and constituting a loop structure, where the nucleomonomers are
selected from the group consisting of G and A.
Similarly, Y may be selected from (a) nothing; (b) an
oligonucleotide of about 1 to about 20 nucleotides covalently
bonded to the 5' end of oligoB and constituting a 5' overhang; (c)
an oligonucleotide of about 1 to about 20 nucleotides covalently
bonded to the 3' end of oligoA and constituting a 3' overhang; (d)
and an oligonucleotide of about 4 to about 20 nucleomonomers
covalently bonded to the 3' end of oligoA and the 5' end of oligoB
and constituting a loop structure, where the nucleomonomers are
selected from the group consisting of G and A.
Similarly, the invention includes a double-stranded oligonucleotide
composition having the structure depicted in FIG. 11: wherein (1)
oligoA is 5'-(N).sub.15-40-(M).sub.2-8-3' and oligoB is
5'-(N).sub.15-40-(M).sub.2-8-3', wherein each of N and M is
independently a nucleomonomer; (2) both of the sequences of Ns are
complementary oligonucleotide strands of equal length having
between about 15 and 40 nucleomonomers; (3) at least one of the
sequences of Ns, optionally with some or all of the flanking Ms,
corresponds to a target gene sequence. Both of the sequences of Ms
are complementary oligonucleotide strands of between about 2 and
about 8 nucleomonomers in length. The two M strands are optionally
of the same length.
The group X indicated by the curved line is selected from (a)
nothing; (b) an oligonucleotide of about 1 to about 20 nucleotides
covalently bonded to the 5' end of oligoA and constituting a 5'
overhang; (c) an oligonucleotide of about 1 to about 20 nucleotides
covalently bonded to the 3' end of oligoB and constituting a 3'
overhang; (d) and an oligonucleotide of about 4 to about 20
nucleomonomers covalently bonded to the 3' end of oligoB and the 5'
end of oligoA and constituting a loop structure, where the
nucleomonomers are selected from the group consisting of G and
A.
Likewise, the invention pertains to a double-stranded
oligonucleotide composition having the structure depicted in FIG.
12, wherein (1) oligoA is 5'-(Z).sub.2-8--(N).sub.12-40-3' and
oligoB is 5'-(Z).sub.2-8--(N).sub.12-40-3', wherein each of N and Z
is independently a nucleomonomer; (2) both of the sequences of Ns
are complementary oligonucleotide strands of equal length having
between about 12 and 40 nucleomonomers; (3) at least one of the
sequences of Ns, optionally with some or all of the flanking Zs,
corresponds to a target gene sequence. Both of the sequences of Zs
are complementary oligonucleotide strands of between about 2 and
about 8 nucleomonomers in length. The two Z strands are optionally
of the same length.
Here, Y is selected from (a) nothing; (b) an oligonucleotide of
about 1 to about 20 nucleotides covalently bonded to the 5' end of
oligoB and constituting a 5' overhang; (c) an oligonucleotide of
about 1 to about 20 nucleotides covalently bonded to the 3' end of
oligoA and constituting a 3' overhang; (d) and an oligonucleotide
of about 4 to about 20 nucleomonomers covalently bonded to the 3'
end of oligoA and the 5' end of oligoB and constituting a loop
structure, where the nucleomonomers are selected from the group
consisting of G and A.
In one embodiment, the double-stranded duplex of an oligonucleotide
of the invention is from between about 12 to about 50
nucleomonomers in length, i.e., the number of nucleotides of the
double-stranded oligonucleotide which hybridize to the
complementary sequence of the double-stranded oligonucleotide to
form the double-stranded duplex structure is from about 12 to about
50 nucleomonomers in length. In another embodiment, the
double-stranded duplex of an oligonucleotide of the invention is
from between about 12 to about 40 nucleomonomers in length.
In one embodiment, the double-stranded duplex of an oligonucleotide
of the invention is at least about 25 nucleomonomers in length. In
one embodiment, the double-stranded duplex is greater than about 25
nucleomonomers in length. In one embodiment, a double-stranded
duplex is at least about 26, 27, 28, 29, 30, at least about 40, at
least about 50, at least about 60, at least about 70, at least
about 80, or at least about 90 nucleomonomers in length. In another
embodiment, the double-stranded duplex is less than about 25
nucleomonomers in length. In one embodiment, a double-stranded
duplex is at least about 10, at least about 15, at least about 20,
at least about 22, at least about 23 or at least about 24
nucleomonomers in length.
In one embodiment, the number of Ns in each strand of the duplex is
about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
or 27. In another embodiment, the number of Ns in each strand of
the duplex is about 30, 35, 40, 45, or 50. In one embodiment, the
number of Ns in each strand of the duplex is about 19. In a
preferred embodiment, the number of Ns in each strand of the duplex
is about 27. In another embodiment, the number of Ns in each strand
of the duplex is about 27 (e.g., is 26, 27, or 28). In another
embodiment, the number of Ns in each strand of the duplex is
27.
In one embodiment, an individual nucleic acid molecule of a
double-stranded oligonucleotide of the invention is at least about
25 nucleomonomers in length. For example, when the double-stranded
oligonucleotide of the invention is comprised of one nucleic acid
molecule, that individual molecule is at least about 25
nucleomonomers in length or when the double-stranded
oligonucleotide of the invention is comprised of two separate
nucleic acid molecules, the length of at least one of the
individual nucleic acid molecules is at least about 25
nucleomonomers in length.
A variety of nucleotides of different lengths may be used. In one
embodiment, an individual nucleic acid molecule comprising a
double-stranded oligonucleotide of the invention is greater than
about 25 nucleomonomers in length. In one embodiment, an individual
nucleic acid molecule comprising a double-stranded oligonucleotide
of the invention is at least about 26, 27, 28, 29, 30, at least
about 40, at least about 50, or at least about 60, at least about
70, at least about 80, or at least about 90 nucleomonomers in
length. In another embodiment, an individual nucleic acid molecule
comprising a double-stranded oligonucleotide of the invention is
less than about 25 nucleomonomers in length. In one embodiment, an
individual nucleic acid molecule comprising a double-stranded
oligonucleotide of the invention is at least about 10, at least
about 15, at least about 20, at least about 22, at least about 23
or at least about 24 nucleomonomers in length.
Double-stranded molecules of the invention may comprise a first
nucleotide sequence which is antisense to at least part of the
target gene and a second nucleotide sequence which is complementary
to the first nucleotide sequence; i.e., is sense to at least part
of the target gene. In one embodiment, the second nucleotide
sequence of the double-stranded molecule comprises a nucleotide
sequence which is at least about 100% complementary to the
antisense molecule.
In another embodiment, the second nucleotide sequence of
double-stranded molecules of the invention may comprise a
nucleotide sequence which is at least about 95% complementary to
the antisense molecule. In another embodiment, the second
nucleotide sequence of double-stranded molecules of the invention
may comprise a nucleotide sequence which is at least about 90%
complementary to the antisense molecule. In another embodiment, the
second nucleotide sequence of double-stranded molecules of the
invention may comprise a nucleotide sequence which is at least
about 80% complementary to the antisense molecule. In another
embodiment, the second nucleotide sequence of double-stranded
molecules of the invention may comprises a nucleotide sequence
which is at least about 60% complementary to the antisense
molecule. In another embodiment, the second nucleotide sequence of
double-stranded molecules of the invention may comprise a
nucleotide sequence which is at least about 100% complementary to
the antisense molecule.
To determine the percent identity of two nucleic acid sequences,
the sequences are aligned for optimal comparison purposes (e.g.,
gaps can be introduced in one or both of a first and a second amino
acid or nucleic acid sequence for optimal alignment and
non-identical sequences can be disregarded for comparison
purposes). When a position in the first sequence is occupied by the
same nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which need
to be introduced for optimal alignment of the two sequences. The
percent complementarity can be determined analogously; when a
position in one sequence occupied by a nucleotide that is
complementary to the nucleotide in the other sequence, then the
molecules are complementary at that position.
The comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical
algorithm. In a preferred embodiment, the percent identity between
two nucleotide sequences is determined using e.g., the GAP program
in the GCG software package, using a NWSgapdna. CMP matrix and a
gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3,
4, 5, or 6. In another embodiment, the percent identity between two
nucleotide sequences is determined using the algorithm of E. Meyers
and W. Miller (Comput. Appl. Biosci. 4:11-17 (1988)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4.
The nucleic acid sequences of the present invention can further be
used as a "query sequence" to perform alignments against sequences
in public databases. Such searches can be performed using the
NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990)
J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be
performed with the NBLAST program, score=100, wordlength=12. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (1997) Nucleic Acids
Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. See, e.g., the NIH website.
In yet another embodiment, a first antisense sequence of the
double-stranded molecule hybridizes to its complementary second
sequence of the double-stranded molecule under stringent
hybridization conditions. As used herein, the term "hybridizes
under stringent conditions" is intended to describe conditions for
hybridization and washing under which nucleotide sequences at least
60% complementary to each other typically remain hybridized to each
other. Preferably, the conditions are such that sequences at least
about 70%, more preferably at least about 80%, even more preferably
at least about 85% or 90% complementary to each other typically
remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting
example of stringent hybridization conditions are hybridization in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50.degree. C., preferably at 55.degree. C., more preferably at
60.degree. C., and even more preferably at 65.degree. C. Ranges
intermediate to the above-recited values, e.g., at 60-65.degree. C.
or at 55-60.degree. C. are also intended to be encompassed by the
present invention. Alternatively, formamide can be included in the
hybridization solution, using methods and conditions also known in
the art.
One of the sequences (or molecules) of the double-stranded
oligonucleotide of the invention is antisense to the target gene.
As used herein, the term "antisense sequence" includes nucleotide
sequences which bind to the "sense" strand of the nucleotide
sequence of the target gene (e.g., polynucleotides such as DNA,
mRNA (including pre-mRNA) molecules). When the antisense sequences
of the invention bind to nucleic acid molecules, they can bind to
any region of a nucleic acid molecule, including e.g., introns,
exons, 5', or 3' untranslated regions. Antisense sequences that
work by binding to a target and activating RNase H preferably bind
within an intron, an exon, the 5' untranslated region, or the 3'
untranslated region of a nucleic acid target molecule.
Preferably, the oligonucleotide compositions of the invention do
not activate the interferon pathway, e.g., as evidenced by the lack
of induction of the double-stranded RNA, interferon-inducible
protein kinase, PKR.
In one embodiment, modifications are made to a double-stranded RNA
molecule which would normally activate the interferon pathway such
that the interferon pathway is not activated. For example, the
interferon pathway is activated by double-stranded unmodified RNA.
The cellular recognition of double-stranded RNA is highly specific
and modifying one or both of the strands of a double-stranded
duplex enables the double-stranded RNA molecule to evade the
double-stranded RNA recognition machinery of the cell but would
still allow for the activation of the RNAi pathway.
The ability of a double-stranded oligonucleotide to activate
interferon could be assessed by testing for expression of the
double-stranded RNA, Interferon-Inducible Protein Kinase, PKR using
techniques known in the art and also testing for the ability of the
double-stranded molecule to effect target gene inhibition.
Accordingly, in one embodiment, the invention provides a method of
testing for the ability of a double-stranded RNA molecule to induce
interferon by testing for the ability of the oligonucleotide to
activate PKR. Compositions that do not activate PKR (i.e., do not
activate the interferon pathway) are then selected for use to
inhibit gene transcription in cells, e.g., in therapeutics or
functional genomics.
Without being limited to any particular mechanism of action, an
antisense sequence used in a double-stranded oligonucleotide
composition of the invention that can specifically hybridize with a
nucleotide sequence within the target gene (i.e., can be
complementary to a nucleotide sequence within the target gene) may
achieve its affects based on, e.g.: (1) binding to target mRNA and
sterically blocking the ribosome complex from translating the mRNA;
(2) binding to target mRNA and triggering mRNA cleavage by RNase H;
(3) binding to double-stranded DNA in the nucleus and forming a
triple helix; (4) hybridizing to open DNA loops created by RNA
polymerase; (5) interfering with mRNA splicing; (6) interfering
with transport of mRNA from the nucleus to the cytoplasm; or (7)
interfering with translation through inhibition of the binding of
initiation factors or assembly of ribosomal subunits (i.e., at the
start codon).
In one embodiment, an antisense sequence of the double-stranded
oligonucleotides of the invention is complementary to a target
nucleic acid sequence over at least about 80% of the length of the
antisense sequence. In another embodiment, the antisense sequence
of the double-stranded oligonucleotide of the invention is
complementary to a target nucleic acid sequence over at least about
90-95% of the length of the antisense sequence. In another
embodiment, the antisense sequence of the double-stranded
oligonucleotide of the invention is complementary to a target
nucleic acid sequence over the entire length of the antisense
sequence.
In yet another embodiment, an antisense sequence of the
double-stranded oligonucleotide hybridizes to at least a portion of
the target gene under stringent hybridization conditions.
In one embodiment, antisense sequences of the invention are
substantially complementary to a target nucleic acid sequence. In
one embodiment, an antisense RNA molecule comprises a nucleotide
sequence which is at least about 100% complementary to a portion of
the target gene. In another embodiment, an antisense RNA molecule
comprises a nucleotide sequence which is at least about 90%
complementary to a portion of the target gene. In another
embodiment, an antisense RNA molecule comprises a nucleotide
sequence which is at least about 80% complementary to a portion of
the target gene. In another embodiment, an antisense RNA molecule
comprises a nucleotide sequence which is at least about 60%
complementary to a portion of the target gene. In another
embodiment, an antisense RNA molecule comprises a nucleotide
sequence which is at least about 100% complementary to a portion of
the target gene. Preferably, no loops greater than about 8
nucleotides are formed by areas of non-complementarity between the
oligonucleotide and the target.
In one embodiment, an antisense nucleotide sequence of the
invention is complementary to a target nucleic acid sequence over
at least about 80% of the length of the antisense sequence. In
another embodiment, an antisense sequence of the invention is
complementary to a target nucleic acid sequence over at least about
90-95% of the length of the antisense sequence. In another
embodiment, an antisense sequence of the invention is complementary
to a target nucleic acid sequence over the entire length of the
antisense sequence.
The antisense sequences used in an oligonucleotide composition of
the invention may be of any type, e.g., including morpholino
oligonucleotides, RNase H activating oligonucleotides, or
ribozymes.
In one embodiment, a double-stranded oligonucleotide of the
invention can comprise (i.e., be a duplex of) one nucleic acid
molecule which is DNA and one nucleic acid molecule which is
RNA.
Antisense sequences of the invention can be "chimeric
oligonucleotides" which comprise an RNA-like and a DNA-like region.
The language "RNase H activating region" includes a region of an
oligonucleotide, e.g., a chimeric oligonucleotide, that is capable
of recruiting RNase H to cleave the target RNA strand to which the
oligonucleotide binds. Typically, the RNase activating region
contains a minimal core (of at least about 3-5, typically between
about 3-12, more typically, between about 5-12, and more preferably
between about 5-10 contiguous nucleomonomers) of DNA or DNA-like
nucleomonomers. (See, e.g., U.S. Pat. No. 5,849,902). Preferably,
the RNase H activating region comprises about nine contiguous
deoxyribose containing nucleomonomers.
In one embodiment, the contiguous nucleomonomers are linked by a
substitute linkage, e.g., a phosphorothioate linkage. In one
embodiment, an antisense sequence of the invention is unstable,
i.e., is degraded in a cell, in the absence of the second strand
(or self complementary sequence) which forms a double-stranded
oligonucleotide of the invention. For example, in one embodiment, a
chimeric antisense sequence comprises unmodified DNA nucleomonomers
in the gap rather than phosphorothioate DNA.
The language "non-activating region" includes a region of an
antisense sequence, e.g., a chimeric oligonucleotide, that does not
recruit or activate RNase H. Preferably, a non-activating region
does not comprise phosphorothioate DNA. The oligonucleotides of the
invention comprise at least one non-activating region. In one
embodiment, the non-activating region can be stabilized against
nucleases or can provide specificity for the target by being
complementary to the target and forming hydrogen bonds with the
target nucleic acid molecule, which is to be bound by the
oligonucleotide.
Antisense sequences of the present invention may include
"morpholino oligonucleotides." Morpholino oligonucleotides are
non-ionic and function by an RNase H-independent mechanism. Each of
the 4 genetic bases (Adenine, Cytosine, Guanine, and
Thymine/Uracil) of the morpholino oligonucleotides is linked to a
6-membered morpholine ring. Morpholino oligonucleotides are made by
joining the 4 different subunit types by, e.g., non-ionic
phosphorodiamidate inter-subunit linkages. An example of a 2
subunit morpholino oligonucleotide is shown in FIG. 13.
Morpholino oligonucleotides have many advantages including:
complete resistance to nucleases (Antisense & Nucl. Acid Drug
Dev. 1996. 6:267); predictable targeting (Biochemica Biophysica
Acta. 1999. 1489:141); reliable activity in cells (Antisense &
Nucl. Acid Drug Dev. 1997. 7:63); excellent sequence specificity
(Antisense & Nucl. Acid Drug Dev. 1997. 7:151); minimal
non-antisense activity (Biochemica Biophysica Acta. 1999.
1489:141); and simple osmotic or scrape delivery (Antisense &
Nucl. Acid Drug Dev. 1997. 7:291). Morpholino oligonucleotides are
also preferred because of their non-toxicity at high doses. A
discussion of the preparation of morpholino oligonucleotides can be
found in Antisense & Nucl. Acid Drug Dev. 1997. 7:187.
Uptake of Oligonucleotides by Cells
Oligonucleotides and oligonucleotide compositions are contacted
with (i.e., brought into contact with, also referred to herein as
administered or delivered to) and taken up by one or more cells or
a cell lysate. The term "cells" includes prokaryotic and eukaryotic
cells, preferably vertebrate cells, and, more preferably, mammalian
cells. In a preferred embodiment, the oligonucleotide compositions
of the invention are contacted with human cells.
Oligonucleotide compositions of the invention can be contacted with
cells in vitro, e.g., in a test tube or culture dish, (and may or
may not be introduced into a subject) or in vivo, e.g., in a
subject such as a mammalian subject. Oligonucleotides are taken up
by cells at a slow rate by endocytosis, but endocytosed
oligonucleotides are generally sequestered and not available, e.g.,
for hybridization to a target nucleic acid molecule. In one
embodiment, cellular uptake can be facilitated by electroporation
or calcium phosphate precipitation. However, these procedures are
only useful for in vitro or ex vivo embodiments, are not convenient
and, in some cases, are associated with cell toxicity.
In another embodiment, delivery of oligonucleotides into cells can
be enhanced by suitable art recognized methods including calcium
phosphate, DMSO, glycerol or dextran, electroporation, or by
transfection, e.g., using cationic, anionic, or neutral lipid
compositions or liposomes using methods known in the art (see e.g.,
WO 90/14074; WO 91/16024; WO 91/17424; U.S. Pat. No. 4,897,355;
Bergan et al. 1993. Nucleic Acids Research. 21:3567). Enhanced
delivery of oligonucleotides can also be mediated by the use of
vectors (See e.g., Shi, Y. 2003. Trends Genet 2003 Jan. 19:9;
Reichhart J M et al. Genesis. 2002. 34(1-2):160-4, Yu et al. 2002.
Proc. Natl. Acad Sci. USA 99:6047; Sui et al. 2002. Proc. Natl.
Acad Sci. USA 99:5515) viruses, polyamine or polycation conjugates
using compounds such as polylysine, protamine, or N1,
N12-bis(ethyl) spermine (see, e.g., Bartzatt, R. et al 1989.
Biotechnol. Appl. Biochem. 11:133; Wagner E. et al. 1992. Proc.
Natl. Acad. Sci. 88:4255).
The optimal protocol for uptake of oligonucleotides will depend
upon a number of factors, the most crucial being the type of cells
that are being used. Other factors that are important in uptake
include, but are not limited to, the nature and concentration of
the oligonucleotide, the confluence of the cells, the type of
culture the cells are in (e.g., a suspension culture or plated) and
the type of media in which the cells are grown. Examples of
different protocols for different cell types are set forth in the
Examples section.
Employing a 1 milliliter final volume solely as a reference volume,
exemplary amounts of reagents which may be used to transfect cells
with nucleic acid molecules of the invention (e.g., double-stranded
oligonucleotides such as, for example, STEALTH.TM. RNA) include the
following. For nucleic acid molecules of the invention (e.g.,
STEALTH.TM. RNA), the amount present may be between about 0.1
picomoles and about 900 nanomoles, between about 0.1 picomoles and
about 700 nanomoles, between about 0.1 picomoles and about 500
nanomoles, between about 0.1 picomoles and about 300 nanomoles,
between about 0.1 picomoles and about 200 nanomoles, between about
0.1 picomoles and about 100 nanomoles, between about 0.1 picomoles
and about 50 nanomoles, between about 0.1 picomoles and about 25
nanomoles, between about 0.1 picomoles and about 1.0 nanomole,
between about 0.1 picomoles and about 800 picomoles, between about
0.1 picomoles and about 600 picomoles, between about 0.1 picomoles
and about 500 picomoles, between about 0.1 picomoles and about 300
picomoles, between about 0.1 picomoles and about 200 picomoles,
between about 1 picomole and about 900 nanomoles, between about 1
and about 600 picomoles, between about 1 and about 500 picomoles,
between about 1 and about 400 picomoles, between about 100 and
about 800 picomoles, between about 200 and about 800 picomoles,
between about 300 and about 800 picomoles, between about 400 and
about 800 picomoles, between about 200 and about 700 picomoles,
between about 50 and about 800 picomoles, between about 50 and
about 500 picomoles, between about 50 and about 400 picomoles,
between about 50 and about 300 picomoles, between about 50 and
about 200 picomoles, between about 100 and about 200 picomoles,
between about 100 and about 300 picomoles, between about 1 nanomole
and about 800 nanomoles, between about 100 nanomoles and about 800
nanomoles, between about 100 nanomoles and about 900 nanomoles,
between about 200 nanomoles and about 900 nanomoles, between about
300 nanomoles and about 900 nanomoles, between about 400 nanomoles
and about 900 nanomoles, or between about 500 nanomoles and about
900 nanomoles.
Further, the total number of cells present may be between about
1.0.times.10.sup.3 and about 1.0.times.10.sup.6, between about
4.0.times.10.sup.3 and about 1.0.times.10.sup.6, between about
5.0.times.10.sup.3 and about 1.0.times.10.sup.6, between about
8.0.times.10.sup.3 and about 1.0.times.10.sup.6, between about
9.0.times.10.sup.3 and about 5.0.times.10.sup.5, between about
1.0.times.10.sup.3 and about 1.0.times.10.sup.5, between about
5.0.times.10.sup.4 and about 1.0.times.10.sup.4, between about
4.0.times.10.sup.3 and about 5.0.times.10.sup.5, or between about
1.0.times.10.sup.4 and about 1.0.times.10.sup.5.
The amount of LIPOFECTAMINE.TM. 2000 or OLIGOFECTAMINE.TM., when
present as a transfection reagent may be between 0.5 nanoliters and
100 microliters, between 5 nanoliters and 100 microliters, between
50 nanoliters and 100 microliters, between 100 nanoliters and 100
microliters, between 200 nanoliters and 100 microliters, between
300 nanoliters and 100 microliters, between 500 nanoliters and 100
microliters, between 750 nanoliters and 100 microliters, between
1.0 microliter and 100 microliters, between 10 microliters and 100
microliters, between 50 microliters and 100 microliters, between
1.0 microliter and 75 microliters, between 1.0 microliter and 50
microliters, or between 1.0 microliters and 30 microliters.
Detectably labeled oligonucleotide controls may be contacted with
cells in concentrations between about 0.1 nanomoles and 1000
nanomoles, between about 1.0 nanomole and 1000 nanomoles, between
about 5.0 nanomoles and 1000 nanomoles, between about 10 nanomoles
and 1000 nanomoles, between about 20 nanomoles and 1000 nanomoles,
between about 40 nanomoles and 1000 nanomoles, between about 60
nanomoles and 1000 nanomoles, between about 100 nanomoles and 1000
nanomoles, between about 0.1 nanomole and 800 nanomoles, between
about 0.1 nanomoles and 700 nanomoles, between about 0.1 nanomoles
and 600 nanomoles, between about 0.1 nanomoles and 500 nanomoles,
between about 0.1 nanomoles and 400 nanomoles, between about 10
nanomoles and 600 nanomoles, between about 10 nanomoles and 500
nanomoles, between about 10 nanomoles and 300 nanomoles, between
about 10 nanomoles and 200 nanomoles, between about 10 nanomoles
and 100 nanomoles, between about 10 nanomoles and 50 nanomoles, or
between about 20 nanomoles and 200 nanomoles.
Exemplary formulations of the above components are set out in the
product literature and table set forth in Example 15.
Conjugating Agents
Conjugating agents bind to the oligonucleotide in a covalent
manner. In one embodiment, oligonucleotides can be derivatized or
chemically modified by binding to a conjugating agent to facilitate
cellular uptake. For example, covalent linkage of a cholesterol
moiety to an oligonucleotide can improve cellular uptake by 5- to
10-fold which in turn improves DNA binding by about 10-fold
(Boutorin et al., 1989, FEBS Letters 254:129-132). Conjugation of
octyl, dodecyl, and octadecyl residues enhances cellular uptake by
3-, 4-, and 10-fold as compared to unmodified oligonucleotides
(Vlassov et al., 1994, Biochimica et Biophysica Acta 1197:95-108).
Similarly, derivatization of oligonucleotides with poly-L-lysine
can aid oligonucleotide uptake by cells (Schell, 1974, Biochem.
Biophys. Acta 340:323, and Lemaitre et al., 1987, Proc. Natl. Acad.
Sci. USA 84:648).
Certain protein carriers can also facilitate cellular uptake of
oligonucleotides, including, for example, serum albumin, nuclear
proteins possessing signals for transport to the nucleus, and viral
or bacterial proteins capable of cell membrane penetration.
Therefore, protein carriers are useful when associated with or
linked to the oligonucleotides. Accordingly, the present invention
provides for derivatization of oligonucleotides with groups capable
of facilitating cellular uptake, including hydrocarbons and
non-polar groups, cholesterol, long chain alcohols (i.e., hexanol),
poly-L-lysine and proteins, as well as other aryl or steroid groups
and polycations having analogous beneficial effects, such as phenyl
or naphthyl groups, quinoline, anthracene or phenanthracene groups,
fatty acids, fatty alcohols and sesquiterpenes, diterpenes, and
steroids. A major advantage of using conjugating agents is to
increase the initial membrane interaction that leads to a greater
cellular accumulation of oligonucleotides.
Encapsulating Agents
Encapsulating agents entrap oligonucleotides within vesicles. In
another embodiment of the invention, an oligonucleotide may be
associated with a carrier or vehicle, e.g., liposomes or micelles,
although other carriers could be used, as would be appreciated by
one skilled in the art. Liposomes are vesicles made of a lipid
bilayer having a structure similar to biological membranes. Such
carriers are used to facilitate the cellular uptake or targeting of
the oligonucleotide, or improve the oligonucleotide's
pharmacokinetic or toxicologic properties.
For example, the oligonucleotides of the present invention may also
be administered encapsulated in liposomes, pharmaceutical
compositions wherein the active ingredient is contained either
dispersed or variously present in corpuscles consisting of aqueous
concentric layers adherent to lipidic layers. The oligonucleotides,
depending upon solubility, may be present both in the aqueous layer
and in the lipidic layer, or in what is generally termed a
liposomic suspension. The hydrophobic layer, generally but not
exclusively, comprises phopholipids such as lecithin and
sphingomyelin, steroids such as cholesterol, more or less ionic
surfactants such as diacetylphosphate, stearylamine, or
phosphatidic acid, or other materials of a hydrophobic nature. The
diameters of the liposomes generally range from about 15 nm to
about 5 microns.
The use of liposomes as drug delivery vehicles offers several
advantages. Liposomes increase intracellular stability, increase
uptake efficiency and improve biological activity. Liposomes are
hollow spherical vesicles composed of lipids arranged in a similar
fashion as those lipids which make up the cell membrane. They have
an internal aqueous space for entrapping water soluble compounds
and range in size from 0.05 to several microns in diameter. Several
studies have shown that liposomes can deliver nucleic acids to
cells and that the nucleic acids remain biologically active. For
example, a liposome delivery vehicle originally designed as a
research tool, such as Lipofectin or LIPOFECTAMINE.TM. 2000, can
deliver intact nucleic acid molecules to cells.
Specific advantages of using liposomes include the following: they
are non-toxic and biodegradable in composition; they display long
circulation half-lives; and recognition molecules can be readily
attached to their surface for targeting to tissues. Finally,
cost-effective manufacture of liposome-based pharmaceuticals,
either in a liquid suspension or lyophilized product, has
demonstrated the viability of this technology as an acceptable drug
delivery system.
Complexing Agents
Complexing agents bind to the oligonucleotides of the invention by
a strong but non-covalent attraction (e.g., an electrostatic, van
der Waals, pi-stacking, etc. interaction). In one embodiment,
oligonucleotides of the invention can be complexed with a
complexing agent to increase cellular uptake of oligonucleotides.
An example of a complexing agent includes cationic lipids. Cationic
lipids can be used to deliver oligonucleotides to cells.
The term "cationic lipid" includes lipids and synthetic lipids
having both polar and non-polar domains and which are capable of
being positively charged at or around physiological pH and which
bind to polyanions, such as nucleic acids, and facilitate the
delivery of nucleic acids into cells. In general cationic lipids
include saturated and unsaturated alkyl and alicyclic ethers and
esters of amines, amides, or derivatives thereof. Straight-chain
and branched alkyl and alkenyl groups of cationic lipids can
contain, e.g., from 1 to about 25 carbon atoms. Preferred straight
chain or branched alkyl or alkene groups have six or more carbon
atoms. Alicyclic groups include cholesterol and other steroid
groups. Cationic lipids can be prepared with a variety of
counterions (anions) including, e.g., Cl.sup.-, Br.sup.-, I.sup.-,
F.sup.-, acetate, trifluoroacetate, sulfate, nitrite, and
nitrate.
Examples of cationic lipids include polyethylenimine,
polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a
combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE.TM.
(e.g., LIPOFECTAMINE.TM. 2000), DOPE, Cytofectin (Gilead Sciences,
Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.).
Exemplary cationic liposomes can be made from
N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride
(DOTMA), N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium
methylsulfate (DOTAP),
3.beta.-[N--(N',N'-dimethylaminoethane)carbamoyl]cholesterol
(DC-Chol),
2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamin-
ium trifluoroacetate (DOSPA),
1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide;
and dimethyldioctadecylammonium bromide (DDAB). The cationic lipid
N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTMA), for example, was found to increase 1000-fold the antisense
effect of a phosphothioate oligonucleotide. (Vlassov et al., 1994,
Biochimica et Biophysica Acta 1197:95-108). Oligonucleotides can
also be complexed with, e.g., poly (L-lysine) or avidin and lipids
may, or may not, be included in this mixture, e.g., steryl-poly
(L-lysine).
Cationic lipids have been used in the art to deliver
oligonucleotides to cells (see, e.g., U.S. Pat. Nos. 5,855,910;
5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al. 1996.
Proc. Natl. Acad. Sci. USA 93:3176; Hope et al. 1998. Molecular
Membrane Biology 15:1). Other lipid compositions which can be used
to facilitate uptake of the instant oligonucleotides can be used in
connection with the claimed methods. In addition to those listed
supra, other lipid compositions are also known in the art and
include, e.g., those taught in U.S. Pat. No. 4,235,871; U.S. Pat.
Nos. 4,501,728; 4,837,028; 4,737,323.
In one embodiment lipid compositions can further comprise agents,
e.g., viral proteins to enhance lipid-mediated transfections of
oligonucleotides (Kamata, et al., 1994. Nucl. Acids. Res. 22:536).
In another embodiment, oligonucleotides are contacted with cells as
part of a composition comprising an oligonucleotide, a peptide, and
a lipid as taught, e.g., in U.S. Pat. No. 5,736,392. Improved
lipids have also been described which are serum resistant (Lewis,
et al., 1996. Proc. Natl. Acad. Sci. 93:3176). Cationic lipids and
other complexing agents act to increase the number of
oligonucleotides carried into the cell through endocytosis.
In another embodiment N-substituted glycine oligonucleotides
(peptoids) can be used to optimize uptake of oligonucleotides.
Peptoids have been used to create cationic lipid-like compounds for
transfection (Murphy, et al., 1998. Proc. Natl. Acad. Sci.
95:1517). Peptoids can be synthesized using standard methods (e.g.,
Zuckermann, R. N., et al. 1992. J. Am. Chem. Soc. 114:10646;
Zuckermann, R. N., et al. 1992. Int. J. Peptide Protein Res.
40:497). Combinations of cationic lipids and peptoids, liptoids,
can also be used to optimize uptake of the subject oligonucleotides
(Hunag, et al., 1998. Chemistry and Biology. 5:345). Liptoids can
be synthesized by elaborating peptoid oligonucleotides and coupling
the amino terminal submonomer to a lipid via its amino group
(Hunag, et al., 1998. Chemistry and Biology. 5:345).
It is known in the art that positively charged amino acids can be
used for creating highly active cationic lipids (Lewis et al. 1996.
Proc. Natl. Acad. Sci. U.S.A. 93:3176). In one embodiment, a
composition for delivering oligonucleotides of the invention
comprises a number of arginine, lysine, histidine or ornithine
residues linked to a lipophilic moiety (see e.g., U.S. Pat. No.
5,777,153).
In another embodiment, a composition for delivering
oligonucleotides of the invention comprises a peptide having from
between about one to about four basic residues. These basic
residues can be located, e.g., on the amino terminal, C-terminal,
or internal region of the peptide. Families of amino acid residues
having similar side chains have been defined in the art. These
families include amino acids with basic side chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine (can
also be considered non-polar), asparagine, glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g.,
threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine, phenylalanine, tryptophan, histidine). Apart from the
basic amino acids, a majority or all of the other residues of the
peptide can be selected from the non-basic amino acids, e.g., amino
acids other than lysine, arginine, or histidine. Preferably a
preponderance of neutral amino acids with long neutral side chains
are used. For example, a peptide such as (N-term)
His-Ile-Trp-Leu-Ile-Tyr-Leu-Trp-Ile-Val-(C-term) (SEQ ID NO: 14)
could be used. In one embodiment such a composition can be mixed
with the fusogenic lipid DOPE as is well known in the art.
In one embodiment, the cells to be contacted with an
oligonucleotide composition of the invention are contacted with a
mixture comprising the oligonucleotide and a mixture comprising a
lipid, e.g., one of the lipids or lipid compositions described
supra for between about 12 hours to about 24 hours. In another
embodiment, the cells to be contacted with an oligonucleotide
composition are contacted with a mixture comprising the
oligonucleotide and a mixture comprising a lipid, e.g., one of the
lipids or lipid compositions described supra for between about 1
and about five days. In one embodiment, the cells are contacted
with a mixture comprising a lipid and the oligonucleotide for
between about three days to as long as about 30 days. In another
embodiment, a mixture comprising a lipid is left in contact with
the cells for at least about five to about 20 days. In another
embodiment, a mixture comprising a lipid is left in contact with
the cells for at least about seven to about 15 days.
For example, in one embodiment, an oligonucleotide composition can
be contacted with cells in the presence of a lipid such as
cytofectin CS or GSV (available from Glen Research; Sterling, Va.),
GS3815, GS2888 for prolonged incubation periods as described
herein.
In one embodiment the incubation of the cells with the mixture
comprising a lipid and an oligonucleotide composition does not
reduce the viability of the cells. Preferably, after the
transfection period the cells are substantially viable. In one
embodiment, after transfection, the cells are between at least
about 70% and at least about 100% viable. In another embodiment,
the cells are between at least about 80% and at least about 95%
viable. In yet another embodiment, the cells are between at least
about 85% and at least about 90% viable.
In one embodiment, oligonucleotides are modified by attaching a
peptide sequence that transports the oligonucleotide into a cell,
referred to herein as a "transporting peptide." In one embodiment,
the composition includes an oligonucleotide which is complementary
to a target nucleic acid molecule encoding the protein, and a
covalently attached transporting peptide.
The language "transporting peptide" includes an amino acid sequence
that facilitates the transport of an oligonucleotide into a cell.
Exemplary peptides which facilitate the transport of the moieties
to which they are linked into cells are known in the art, and
include, e.g., HIV TAT transcription factor, lactoferrin, Herpes
VP22 protein, and fibroblast growth factor 2 (Pooga et al. 1998.
Nature Biotechnology. 16:857; and Derossi et al. 1998. Trends in
Cell Biology. 8:84; Elliott and O'Hare. 1997. Cell 88:223).
For example, in one embodiment, the transporting peptide comprises
an amino acid sequence derived from the antennapedia protein.
Preferably, the peptide comprises amino acids 43-58 of the
antennapedia protein
(Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys)
(SEQ ID NO: 15) or a portion or variant thereof that facilitates
transport of an oligonucleotide into a cell (see, e.g., WO 91/1898;
Derossi et al. 1998. Trends Cell Biol. 8:84). Exemplary variants
are shown in Derossi et al., supra.
In one embodiment, the transporting peptide comprises an amino acid
sequence derived from the transportan, galanin
(1-12)-Lys-mastoparan (1-14) amide, protein. (Pooga et al. 1998.
Nature Biotechnology 16:857). Preferably, the peptide comprises the
amino acids of the transportan protein shown in the sequence
GWTLNSAGYLLGKINLKAL-AALAKKIL (SEQ ID NO: 16) or a portion or
variant thereof that facilitates transport of an oligonucleotide
into a cell.
In one embodiment, the transporting peptide comprises an amino acid
sequence derived from the HIV TAT protein. Preferably, the peptide
comprises amino acids 37-72 of the HIV TAT protein, e.g., shown in
the sequence C(Acm)FITKALGISYGRKKRRQRRR-PPQC (SEQ ID NO: 17) (TAT
37-60; where C(Acm) is Cys-acetamidomethyl) or a portion or variant
thereof, e.g., C(Acm)GRKKRRQRRRPPQC (SEQ ID NO: 18) (TAT 48-40) or
C(Acm)LGISYGRKKRRQRRPPQC (SEQ ID NO: 19) (TAT 43-60) that
facilitates transport of an oligonucleotide into a cell (Vives et
al. 1997. J. Biol. Chem. 272:16010). In another embodiment the
peptide (G)CFITKALGISYGRKKRRQR-RRPPQGSQTHQVSLSKQ (SEQ ID NO: 20)
can be used.
Portions or variants of transporting peptides can be readily tested
to determine whether they are equivalent to these peptide portions
by comparing their activity to the activity of the native peptide,
e.g., their ability to transport fluorescently-labeled
oligonucleotides to cells. Fragments or variants that retain the
ability of the native transporting peptide to transport an
oligonucleotide into a cell are functionally equivalent and can be
substituted for the native peptides.
Oligonucleotides can be attached to the transporting peptide using
known techniques, e.g., (Prochiantz, A. 1996. Curr. Opin.
Neurobiol. 6:629; Derossi et al. 1998. Trends Cell Biol. 8:84; Troy
et al. 1996. J. Neurosci. 16:253), Vives et al. 1997. J. Biol.
Chem. 272:16010). For example, in one embodiment, oligonucleotides
bearing an activated thiol group are linked via that thiol group to
a cysteine present in a transport peptide (e.g., to the cysteine
present in the .beta. turn between the second and the third helix
of the antennapedia homeodomain as taught, e.g., in Derossi et al.
1998. Trends Cell Biol. 8:84; Prochiantz. 1996. Current Opinion in
Neurobiol. 6:629; Allinquant et al. 1995. J. Cell Biol. 128:919).
In another embodiment, a Boc-Cys-(Npys)OH group can be coupled to
the transport peptide as the last (N-terminal) amino acid and an
oligonucleotide bearing an SH group can be coupled to the peptide
(Troy et al. 1996. J. Neurosci. 16:253).
In one embodiment, a linking group can be attached to a
nucleomonomer and the transporting peptide can be covalently
attached to the linker. In one embodiment, a linker can function as
both an attachment site for a transporting peptide and can provide
stability against nucleases. Examples of suitable linkers include
substituted or unsubstituted C.sub.1-C.sub.20 alkyl chains,
C.sub.2-C.sub.20 alkenyl chains, C.sub.2-C.sub.20 alkynyl chains,
peptides, and heteroatoms (e.g., S, O, NH, etc.). Other exemplary
linkers include bifunctional crosslinking agents such as
sulfosuccinimidyl-4-(maleimidophenyl)-butyrate (SMPB) (see, e.g.,
Smith et al. Biochem J 1991. 276: 417-2).
In one embodiment, oligonucleotides of the invention are
synthesized as molecular conjugates which utilize receptor-mediated
endocytotic mechanisms for delivering genes into cells (see, e.g.,
Bunnell et al. 1992. Somatic Cell and Molecular Genetics. 18:559,
and the references cited therein).
Targeting Agents
The delivery of oligonucleotides can also be improved by targeting
the oligonucleotides to a cellular receptor. The targeting moieties
can be conjugated to the oligonucleotides or attached to a carrier
group (i.e., poly(L-lysine) or liposomes) linked to the
oligonucleotides. This method is well suited to cells that display
specific receptor-mediated endocytosis.
For instance, oligonucleotide conjugates to 6-phosphomannosylated
proteins are internalized 20-fold more efficiently by cells
expressing mannose 6-phosphate specific receptors than free
oligonucleotides. The oligonucleotides may also be coupled to a
ligand for a cellular receptor using a biodegradable linker. In
another example, the delivery construct is mannosylated
streptavidin which forms a tight complex with biotinylated
oligonucleotides. Mannosylated streptavidin was found to increase
20-fold the internalization of biotinylated oligonucleotides.
(Vlassov et al. 1994. Biochimica et Biophysica Acta
1197:95-108).
In addition specific ligands can be conjugated to the polylysine
component of polylysine-based delivery systems. For example,
transferrin-polylysine, adenovirus-polylysine, and influenza virus
hemagglutinin HA-2 N-terminal fusogenic peptides-polylysine
conjugates greatly enhance receptor-mediated DNA delivery in
eucaryotic cells. Mannosylated glycoprotein conjugated to
poly(L-lysine) in aveolar macrophages has been employed to enhance
the cellular uptake of oligonucleotides. Liang et al. 1999.
Pharmazie 54:559-566.
Because malignant cells have an increased need for essential
nutrients such as folic acid and transferrin, these nutrients can
be used to target oligonucleotides to cancerous cells. For example,
when folic acid is linked to poly(L-lysine) enhanced
oligonucleotide uptake is seen in promyelocytic leukaemia (HL-60)
cells and human melanoma (M-14) cells. Ginobbi et al. 1997.
Anticancer Res. 17:29. In another example, liposomes coated with
maleylated bovine serum albumin, folic acid, or ferric
protoporphyrin IX, show enhanced cellular uptake of
oligonucleotides in murine macrophages, KB cells, and 2.2.15 human
hepatoma cells. Liang et al. 1999. Pharmazie 54:559-566.
Liposomes naturally accumulate in the liver, spleen, and
reticuloendothelial system (so-called, passive targeting). By
coupling liposomes to various ligands such as antibodies are
protein A, they can be actively targeted to specific cell
populations. For example, protein A-bearing liposomes may be
pretreated with H-2K specific antibodies which are targeted to the
mouse major histocompatibility complex-encoded H-2K protein
expressed on L cells. (Vlassov et al. 1994. Biochimica et
Biophysica Acta 1197:95-108).
Assays of Oligonucleotide Stability
Preferably, the double-stranded oligonucleotides of the invention
are stabilized, i.e., substantially resistant to endonuclease and
exonuclease degradation. An oligonucleotide is defined as being
substantially resistant to nucleases when it is at least about
3-fold more resistant to attack by an endogenous cellular nuclease,
and is highly nuclease resistant when it is at least about 6-fold
more resistant than a corresponding, single-stranded
oligonucleotide. This can be demonstrated by showing that the
oligonucleotides of the invention are substantially resistant to
nucleases using techniques which are known in the art.
One way in which substantial stability can be demonstrated is by
showing that the oligonucleotides of the invention function when
delivered to a cell, e.g., that they reduce transcription or
translation of target nucleic acid molecules, e.g., by measuring
protein levels or by measuring cleavage of mRNA. Assays which
measure the stability of target RNA can be performed at about 24
hours post-transfection (e.g., using Northern blot techniques,
RNase Protection Assays, or QC-PCR assays as known in the art).
Alternatively, levels of the target protein can be measured.
Preferably, in addition to testing the RNA or protein levels of
interest, the RNA or protein levels of a control, non-targeted gene
will be measured (e.g., actin, or preferably a control with
sequence similarity to the target) as a specificity control. RNA or
protein measurements can be made using any art-recognized
technique. Preferably, measurements will be made beginning at about
16-24 hours post transfection. (M. Y. Chiang, et al. 1991. J Biol
Chem. 266:18162-71; T. Fisher, et al. 1993. Nucleic Acids Research.
21 3857).
The ability of an oligonucleotide composition of the invention to
inhibit protein synthesis can be measured using techniques which
are known in the art, for example, by detecting an inhibition in
gene transcription or protein synthesis. For example, Nuclease S1
mapping can be performed. In another example, Northern blot
analysis can be used to measure the presence of RNA encoding a
particular protein. For example, total RNA can be prepared over a
cesium chloride cushion (see, e.g., Ausebel et al., 1987. Current
Protocols in Molecular Biology (Greene & Wiley, New York)).
Northern blots can then be made using the RNA and probed (see,
e.g., Id.). In another example, the level of the specific mRNA
produced by the target protein can be measured, e.g., using PCR. In
yet another example, Western blots can be used to measure the
amount of target protein present. In still another embodiment, a
phenotype influenced by the amount of the protein can be detected.
Techniques for performing Western blots are well known in the art,
see, e.g., Chen et al. J. Biol. Chem. 271:28259.
In another example, the promoter sequence of a target gene can be
linked to a reporter gene and reporter gene transcription (e.g., as
described in more detail below) can be monitored. Alternatively,
oligonucleotide compositions that do not target a promoter can be
identified by fusing a portion of the target nucleic acid molecule
with a reporter gene so that the reporter gene is transcribed. By
monitoring a change in the expression of the reporter gene in the
presence of the oligonucleotide composition, it is possible to
determine the effectiveness of the oligonucleotide composition in
inhibiting the expression of the reporter gene. For example, in one
embodiment, an effective oligonucleotide composition will reduce
the expression of the reporter gene.
A "reporter gene" is a nucleic acid that expresses a detectable
gene product, which may be RNA or protein. Detection of mRNA
expression may be accomplished by Northern blotting and detection
of protein may be accomplished by staining with antibodies specific
to the protein. Preferred reporter genes produce a readily
detectable product. A reporter gene may be operably linked with a
regulatory DNA sequence such that detection of the reporter gene
product provides a measure of the transcriptional activity of the
regulatory sequence. In preferred embodiments, the gene product of
the reporter gene is detected by an intrinsic activity associated
with that product. For instance, the reporter gene may encode a
gene product that, by enzymatic activity, gives rise to a
detectable signal based on color, fluorescence, or luminescence.
Examples of reporter genes include, but are not limited to, those
coding for chloramphenicol acetyl transferase (CAT), luciferase,
.beta.-galactosidase, and alkaline phosphatase.
One skilled in the art would readily recognize numerous reporter
genes suitable for use in the present invention. These include, but
are not limited to, chloramphenicol acetyltransferase (CAT),
luciferase, human growth hormone (hGH), and beta-galactosidase.
Examples of such reporter genes can be found in F. A. Ausubel et
al., Eds., Current Protocols in Molecular Biology, John Wiley &
Sons, New York, (1989). Any gene that encodes a detectable product,
e.g., any product having detectable enzymatic activity or against
which a specific antibody can be raised, can be used as a reporter
gene in the present methods.
One reporter gene system is the firefly luciferase reporter system.
(Gould, S. J., and Subramani, S. 1988. Anal. Biochem., 7:404-408
incorporated herein by reference). The luciferase assay is fast and
sensitive. In this assay, a lysate of the test cell is prepared and
combined with ATP and the substrate luciferin. The encoded enzyme
luciferase catalyzes a rapid, ATP dependent oxidation of the
substrate to generate a light-emitting product. The total light
output is measured and is proportional to the amount of luciferase
present over a wide range of enzyme concentrations.
CAT is another frequently used reporter gene system; a major
advantage of this system is that it has been an extensively
validated and is widely accepted as a measure of promoter activity.
(Gorman C. M., Moffat, L. F., and Howard, B. H. 1982. Mol. Cell.
Biol., 2:1044-1051). In this system, test cells are transfected
with CAT expression vectors and incubated with the candidate
substance within 2-3 days of the initial transfection. Thereafter,
cell extracts are prepared. The extracts are incubated with acetyl
CoA and radioactive chloramphenicol. Following the incubation,
acetylated chloramphenicol is separated from nonacetylated form by
thin layer chromatography. In this assay, the degree of acetylation
reflects the CAT gene activity with the particular promoter.
Another suitable reporter gene system is based on immunologic
detection of hGH. This system is also quick and easy to use.
(Selden, R., Burke-Howie, K. Rowe, M. E., Goodman, H. M., and
Moore, D. D. (1986), Mol. Cell, Biol., 6:3173-3179 incorporated
herein by reference). The hGH system is advantageous in that the
expressed hGH polypeptide is assayed in the media, rather than in a
cell extract. Thus, this system does not require the destruction of
the test cells. It will be appreciated that the principle of this
reporter gene system is not limited to hGH but rather adapted for
use with any polypeptide for which an antibody of acceptable
specificity is available or can be prepared.
In one embodiment, nuclease stability of a double-stranded
oligonucleotide of the invention is measured and compared to a
control, e.g., an RNAi molecule typically used in the art (e.g., a
duplex oligonucleotide of less than 25 nucleotides in length and
comprising 2 nucleotide base overhangs) or an unmodified RNA duplex
with blunt ends.
Monitoring the Effects of Oligonucleotide
Monitoring the effects of double-stranded oligonucleotides of the
invention on cell lines can by performed by the addition of stain
compounds to cells, tissues, or organisms undergoing experiments or
treatment. Addition of a stain compound to cells, tissues, or
organisms in an experiment allows for the monitoring of the effects
of the oligonucleotide at one or more discrete time points or in
real time with continuous monitoring. Effects monitored can include
apoptosis, cellular health and vitality, cell proliferation,
cellular phenotypic changes, and so on. Stain compounds can be
fluorescent or otherwise cause a detectable signal when interacting
with a target.
Methods for monitoring the effects of an oligonucleotide of the
present invention generally comprise contacting one or more cells
with an oligonucleotide molecule and a stain compound, and
detecting a signal from the cells. The contacting step can occur in
one step, where the oligonucleotide molecule and the stain compound
are introduced into the cell simultaneously. Alternatively, the
contacting step can be performed stepwise, where the stain molecule
is introduced into the cell, and then the oligonucleotide molecule
is introduced into the cell, or vise versa. The contacting step can
include the addition of cellular uptake agents such as a
surfactant. Multiple oligonucleotide molecules can be used, such as
2, 3, 4, 5, 6, and so on. Multiple stain compounds can be used,
such as 2, 3, 4, 5, 6, and so on.
Stain compounds can generally be any compound that generates a
detectable signal upon interaction with a target. Compounds
typically are luminescent (e.g. fluorescent, chemiluminescent, or
phosphorescent). Stain compounds can generate the detectable signal
directly (i.e. signal is generated upon interaction), or indirectly
by including a third compound (e.g. the stain compound can be an
enzyme that acts upon a substrate that becomes fluorescent).
Examples of stain compounds include DNA labeled with a fluorescent
molecule, RNA labeled with a fluorescent molecule, an antibody
labeled with a fluorescent molecule, a Fab fragment labeled with a
fluorescent molecule, and so on. The fluorescent molecule can be an
organic compound, or a protein such as green fluorescent protein
(GFP). Antibodies can be a labeled primary antibody, or a
combination of an unlabeled primary antibody and a labeled
secondary antibody. Antibodies can be labeled with a fluorescent or
other detectable group, or can be labeled with an enzyme (such as a
peroxidase, alkaline phosphatase, galactosidase, luciferase, or
lactamase) that can react with a substrate. Stain compounds can
interact with various targets. Targets include nucleic acid (e.g.
DNA), proteins, peptides, and lipids. Targets can also include
cellular structures such as cytoplasm, cytoskeleton, endoplasmic
reticulum (ER), golgi, lysosomes, mitochondria, nucleus, nucleoli,
peroxisomes, and plasma membrane.
Specific examples of stain compounds useful for studying changes in
cell structure include 4',6-diamidino-2-phenylindole
dihydrochloride (useful counterstain for nucleus and chromosomes),
Hoechst 33342 trihydrochloride trihydrate (useful cell-permeant
nuclear counterstain that emits blue fluorescence when bound to
dsDNA; can be used to distinguish condensed pycnotic nuclei in
apoptotic cells and for cell-cycle studies with BrdU), SYTOX Blue
(a blue-fluorescent nuclear and chromosome counterstain that is
impermeant to live cells), SYTOX Green (a green-fluorescent nuclear
and chromosome counterstain that is impermeant to live cells),
YO-PRO-1 iodide (a carbocyanine nucleic acid stain useful for
identifying apoptotic cells), BO-PRO-1 iodide (a carbocyanine
nucleic acid stain), SYTO 59 (a cell-permeant nucleic acid stain),
and TO-PRO-3 iodide (a carbocyanine monomer useful as a dead cell
indicator).
Specific examples of stain compounds useful for studying DNA
fragmentation include 5-bromo-2'-deoxyuridine 5'-triphosphate
(BrdUTP) with Alexa Fluor 488 anti-BrdU, anti-bromodeoxyuridine,
mouse IgG1, monoclonal PRB-1 Alexa Fluor 488 conjugate (anti-BrdU,
Alexa Fluor 488 conjugate), and anti-bromodeoxyuridine, mouse IgG1,
monoclonal PRB-1 Alexa Fluor 594 conjugate (anti-BrdU, Alexa Fluor
594 conjugate).
Specific examples of stain compounds useful for studying cell
proliferation include CyQUANT, and carboxyfluorescein diacetate
succinimidyl ester.
Specific examples of stain compounds useful for studying apoptosis
include caspase substrates rhodamine 110,
bis-(N-CBZ-L-isoleucyl-L-glutamyl-L-threonyl-L-aspartic acid
amide), Z-DEVD-AMC (C.sub.36H.sub.41N.sub.5O.sub.14), Z-DEVD-AMC
(C.sub.36H.sub.41N.sub.5O.sub.14), and rhodamine 110,
bis-(L-aspartic acid amide). Compounds useful for studying
phosphatidylserine exposure include recombinant annexin V
conjugated to green-fluorescent Alexa Fluor 488 dye,
green-fluorescent Alexa Fluor 488 annexin with red-fluorescent
propidium iodide nucleic acid stain, Alexa Fluor 488 annexin V
conjugate with SYTOX Green nucleic acid stain and
C.sub.12-resazurin, recombinant annexin V conjugated to
allophycocyanin (APC) and a SYTOX Green nucleic acid stain,
recombinant annexin V conjugated to R-phycoerythrin (R-PE) and a
SYTOX Green nucleic acid stain, Alexa Fluor 568 annexin V
conjugate, Alexa Fluor 594 annexin V conjugate, Alexa Fluor 350
annexin V conjugate, and Alexa Fluor 647 annexin V conjugate.
Specific examples of stain compounds useful for studying changes in
mitochondria include MitoTracker Red CMXRos (a red-fluorescent dye
that stains mitochondria in live cells), MitoTracker Green FM (a
green-fluorescent mitochondrial stain that stains live cells), and
MitoTracker Orange CMTMRos (an orange-fluorescent mitochondrial
stain that stains live cells).
Specific examples of stain compounds useful for studying changes in
lysosomes include LysoTracker Red DND-99 (a red-fluorescent dye
that stains acidic compartments in live cells), LysoTracker Green
DND-26 (a green-fluorescent dye that stains acidic compartments in
live cells), and LysoSensor Yellow/Blue DND-160 (an acidotropic
probe that accumulates in acidic organelles due to
protonation).
Specific examples of stain compounds useful for studying changes in
plasma membranes include FM 1-43FX (a membrane probe analog
modified to contain an aliphatic amine), Vybrant DiI (a lipophilic
membrane stain), Vybrant DiO (a lipophilic membrane stain), and
Vybrant DiD (a lipophilic membrane stain).
Specific examples of stain compounds useful for studying changes in
the cytoplasm include Calcein AM (a cell-permeant dye used to
determine cell viability in eukaryotic cells), CellTracker Green
CMFDA (a fluorescent chloromethyl derivative that exhibits green
fluorescence in the cytoplasm at physiological pH), and CellTracker
Red CMTPX (a fluorescent chloromethyl derivative that exhibits red
fluorescence in the cytoplasm at physiological pH).
Specific examples of stain compounds useful for studying changes in
the endoplasmic reticulum include ER-Tracker Blue-White DPX (a
photostable probe that is selective for the endoplasmic reticulum
in live cells), SelectFX Alexa Fluor 488 (primary and secondary
antibody pair), and brefeldin A BODIPY 558/568 conjugate (a labeled
reversible inhibitor of protein transport from the endoplasmic
reticulum to the Golgi apparatus).
Specific examples of stain compounds useful for studying changes in
the peroxisome include SelectFX Alexa Fluor 488 (primary and
secondary antibody pair directed against peroxisomal membrane
protein 70).
Specific examples of stain compounds useful for studying changes in
the Golgi include anti-golgin-97 (human) mouse IgG1 monoclonal CDF4
(anti-Golgin-97 antibody), NBD C6-ceramide complexed to BSA, BODIPY
FL C5-ceramide complexed to BSA, and BODIPY TR C5-ceramide
complexed to BSA (fluorescent ceramides are markers for Golgi
Complex in living cells).
Specific examples of stain compounds useful for studying changes in
the nucleoli include SYTO RNASelect green fluorescent cell stain (a
cell permeant nucleic acid stain selective for RNA).
Specific examples of stain compounds useful for studying changes in
the cytoskeleton include Alexa Fluor 488 phalloidin (a
high-affinity probe for F-actin conjugated to a green-fluorescent
dye), rhodamine phalloidin (a high-affinity probe for F-actin
conjugated to the orange-fluorescent dye tetramethylrhodamine
(TRITC)), jasplakinolide (a macrocyclic peptide), latrunculin A
(binding to monomeric G-actin in a 1:1 complex), Oregon Green 488
Taxol (paclitaxel labeled at the 7-carbon with a fluorescent dye),
anti-alpha tubulin mouse IgG1 (enables visualization of
microtubules with an anti-mouse IgG secondary immunoreagent).
Specific examples of stain compounds useful for studying changes in
lipid rafts include Vybrant Alexa Fluor 488 (chlorea toxin subunit
B labeled with a fluorescent dye), Vybrant Alexa Fluor 555 (chlorea
toxin subunit B labeled with a fluorescent dye), and Vybrant Alexa
Fluor 594 (chlorea toxin subunit B labeled with a fluorescent
dye).
Specific examples of stain compounds useful for studying changes in
calcium levels include fluo-4 AM, fura-2 AM, indo-1 AM, and rhod-2
AM.
Specific examples of stain compounds useful for studying changes in
reactive oxygen species levels include
5-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl
ester (a cell permeant indicator for reactive oxygen species that
is nonfluorescent until removal of the acetate groups by
intracellular esterases and oxidation),
6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl
ester (a cell permeant indicator for reactive oxygen species that
is nonfluorescent until removal of the acetate groups by
intracellular esterases and oxidation), dihydroethidium
(hydroethidine) (cell permeant stain becomes red-fluorescent
ethidium and accumulates in the nucleus), aminophenyl fluorescein,
hydroxyphenyl fluorescein, BODIPY 581/591 fatty acid C11, and
glutathione ethyl ester biotin amide.
Specific examples of stain compounds useful for studying changes in
reactive nitrogen species levels include anti-nitrotyrosine rabbit
IgG (used in conjunction with an anti-rabbit IgG secondary
immunoreagent), and DAF-FM diacetate (useful for quantitating low
concentrations of nitric oxide in cells).
Specific examples of stain compounds useful for studying changes in
sodium levels include CoroNa Green AM (exhibits increased green
fluorescence emission upon binding sodium).
Specific examples of stain compounds useful for studying changes in
pH levels include BCECF AM, 5-chloromethyl SNARF-1 acetate, and
6-chloromethyl SNARF-1 acetate.
Specific examples of stain compounds useful for studying changes in
zinc levels include FluoZin-3 tetrapotassium salt (green
fluorescent, suitable for detection of Zn.sup.2+ at 1-100 mM
concentrations), and RhodZin-3 AM (orange-red fluorescent indicator
useful for measuring Zn.sup.2+ in mitochondria).
The contacting step can be performed in a variety of environments
or containers. For example, cells in a centrifuge tube, microscope
slide, or multiwell plate can be contacted. Alternatively, tissues,
tissue slices, or whole organisms can be contacted.
The method can further comprise an incubation step prior to the
detection step. The incubation step can vary in length depending on
the oligonucleotide experiment.
The detecting step can generally be performed by any machine or
method suitable for detecting the signal. Examples of detecting
steps include use of a fluorescence microscope, use of a plate
reader, and use of a flow cytometer. The detecting step can be
qualitative or quantitative. The detecting step can be performed at
one or more discrete time points, or can be done continuously in
real time.
The detecting step can comprise applying light at an absorbance
wavelength to the cells, and detecting light emitted at a different
wavelength. Examples of pairs of absorbance and emission
wavelengths (in nm) include 346/442, 402/421, 495/519, 555/565,
578/603, 590/617, 650/668, 663/690, 679/702, and 749/775.
The methods can further comprise comparing the detected signal with
signal(s) detected from control samples. For example, a control
cell or cells can be treated with the same stain compound, but not
with the oligonucleotide molecule.
Cellular effects may be exhibited as graded responses (as compared
to all-or-none responses) in individual cells and may effect
different cells in a population differently. Even in cells which
share a common clonal origin, effects may be exhibited in certain
cells in a population but not in others. Thus, methods disclosed
herein may be used to determine the physiological status or
condition of multiple cells in a population and compare that status
or condition of those cells to the status or condition of cells
which have not been treated.
The one or more cells can generally be any type of cell. Examples
of cells include bacterial cells, fungal cells, insect cells, and
mammalian cells. The cells can be a homogeneous or heterogeneous
population. The cells can be mixtures of multiple types of cells
from the same organism, or mixtures of cells from different
organisms. The cells can be "wild-type" or modified through genetic
engineering, viral or pathogen infection, randomly or specifically
mutagenized, and so on.
The effects of the oligonucleotide monitored can generally be any
effect. Effects can include cell viability, cell vitality,
apoptosis, cell proliferation, signal transduction, energy charge,
cell morphology, the activity of one or more enzymes, membrane
potential, gene expression efficiency, cytoskeletal integrity, and
the presence or absence of vacuoles. Effects can be measured
relative to a control cell of the same type that is not treated
with the oligonucleotide molecule. Depending on the effect
measured, the signal obtained from the treated cell may be higher
or lower than the signal obtained from the control cell. The signal
obtained from the treated cell may increase or decrease over time,
depending on the effect of the oligonucleotide molecule. Effects
can include changes in pH, changes in concentration of a material
(e.g. calcium or sodium), changes in shape, changes in oxidation
state, and so on.
Additional embodiments involve kits useful for conducting
oligonucleotide experiments. The kits can comprise one or more of
the oligonucleotides of the invention, and one or more of the above
described stain compounds. The kits can further comprise an
instruction protocol. The kits can comprise one or more containers
for holding the oligonucleotide molecule, the stain compound, or
both. The kits can comprise one or more containers for conducting
the oligonucleotide experiments. The kits can comprise one or more
buffers. The kits can comprise one or more solvents. The kits can
comprise one or more positive or negative standards. The kits can
comprise positive or negative control samples or reference
samples.
Oligonucleotide Synthesis
Oligonucleotides of the invention can be synthesized by any method
known in the art, e.g., using enzymatic synthesis and chemical
synthesis. The oligonucleotides can be synthesized in vitro (e.g.,
using enzymatic synthesis and chemical synthesis) or in vivo (using
recombinant DNA technology well known in the art).
In a preferred embodiment, chemical synthesis is used. Chemical
synthesis of linear oligonucleotides is well known in the art and
can be achieved by solution or solid phase techniques. Preferably,
synthesis is by solid phase methods. Oligonucleotides can be made
by any of several different synthetic procedures including the
phosphoramidite, phosphite triester, H-phosphonate, and
phosphotriester methods, typically by automated synthesis
methods.
Oligonucleotide synthesis protocols are well known in the art and
can be found, e.g., in U.S. Pat. No. 5,830,653; WO 98/13526; Stec
et al. 1984. J. Am. Chem. Soc. 106:6077; Stec et al. 1985. J. Org.
Chem. 50:3908; Stec et al. J. Chromatog. 1985. 326:263; LaPlanche
et al. 1986. Nucl. Acid. Res. 1986. 14:9081; Fasman G. D., 1989.
Practical Handbook of Biochemistry and Molecular Biology. 1989. CRC
Press, Boca Raton, Fla.; Lamone. 1993. Biochem. Soc. Trans. 21:1;
U.S. Pat. No. 5,013,830; U.S. Pat. No. 5,214,135; U.S. Pat. No.
5,525,719; Kawasaki et al. 1993. J. Med. Chem. 36:831; WO 92/03568;
U.S. Pat. No. 5,276,019; and U.S. Pat. No. 5,264,423.
The synthesis method selected can depend on the length of the
desired oligonucleotide and such choice is within the skill of the
ordinary artisan. For example, the phosphoramidite and phosphite
triester method can produce oligonucleotides having 175 or more
nucleotides while the H-phosphonate method works well for
oligonucleotides of less than 100 nucleotides. If modified bases
are incorporated into the oligonucleotide, and particularly if
modified phosphodiester linkages are used, then the synthetic
procedures are altered as needed according to known procedures. In
this regard, Uhlmann et al. (1990, Chemical Reviews 90:543-584)
provide references and outline procedures for making
oligonucleotides with modified bases and modified phosphodiester
linkages. Other exemplary methods for making oligonucleotides are
taught in Sonveaux. 1994. "Protecting Groups in Oligonucleotide
Synthesis"; Agrawal. Methods in Molecular Biology 26:1. Exemplary
synthesis methods are also taught in "Oligonucleotide Synthesis--A
Practical Approach" (Gait, M. J. IRL Press at Oxford University
Press. 1984). Moreover, linear oligonucleotides of defined
sequence, including some sequences with modified nucleotides, are
readily available from several commercial sources.
The oligonucleotides may be purified by polyacrylamide gel
electrophoresis, or by any of a number of chromatographic methods,
including gel chromatography and high pressure liquid
chromatography. To confirm a nucleotide sequence, oligonucleotides
may be subjected to DNA sequencing by any of the known procedures,
including Maxam and Gilbert sequencing, Sanger sequencing,
capillary electrophoresis sequencing, the wandering spot sequencing
procedure or by using selective chemical degradation of
oligonucleotides bound to Hybond paper. Sequences of short
oligonucleotides can also be analyzed by laser desorption mass
spectroscopy or by fast atom bombardment (McNeal, et al., 1982, J.
Am. Chem. Soc. 104:976; Viari, et al., 1987, Biomed. Environ. Mass
Spectrom. 14:83; Grotjahn et al., 1982, Nucl. Acid Res. 10:4671).
Sequencing methods are also available for RNA oligonucleotides.
The quality of oligonucleotides synthesized can be verified by
testing the oligonucleotide by capillary electrophoresis and
denaturing strong anion HPLC (SAX-HPLC) using, e.g., the method of
Bergot and Egan. 1992. J. Chrom. 599:35.
Other exemplary synthesis techniques are well known in the art
(see, e.g., Sambrook et al., Molecular Cloning: a Laboratory
Manual, Second Edition (1989); DNA Cloning, Volumes I and II (D N
Glover Ed. 1985); Oligonucleotide Synthesis (M J Gait Ed, 1984;
Nucleic Acid Hybridisation (B D Hames and S J Higgins eds. 1984); A
Practical Guide to Molecular Cloning (1984); or the series, Methods
in Enzymology (Academic Press, Inc.)).
Uses of Oligonucleotides
The invention also features methods of inhibiting expression of a
protein in a cell including contacting the cell with one of the
above-described oligonucleotide compositions.
The oligonucleotides of the invention can be used in a variety of
in vitro and in vivo situations to specifically inhibit protein
expression. The instant methods and compositions are suitable for
both in vitro and in vivo use.
Methods of the invention may be used for determining the function
of a gene in a cell or an organism or for modulating the function
of a gene in a cell or an organism, being capable of responding to
or mediating RNA interference. The cell is preferably a eukaryotic
cell or a cell line, e.g., an animal cell such as a mammalian cell,
e.g., an embryonic cell, a pluripotent stem cell, a tumor cell,
e.g., a teratocarcinoma cell, or a virus-infected cell. The
organism is preferably a eukaryotic organism, e.g., an animal such
as a mammal, particularly a human.
The invention includes methods to inhibit expression of a target
gene in a cell in vitro. For example, such methods may include
introduction of RNA into a cell in an amount sufficient to inhibit
expression of the target gene, where the RNA is a double-stranded
molecule of the invention. By way of a further example, such an RNA
molecule may have a first strand consisting essentially of a
ribonucleotide sequence that corresponds to a nucleotide sequence
of the target gene, and a second strand consisting essentially of a
ribonucleotide sequence that is complementary to the nucleotide
sequence of the target gene, in which the first and the second
strands are separate complementary strands or are joined by a loop,
and they hybridize to each other to form said double-stranded
molecule, such that the duplex composition inhibits expression of
the target gene. The duplex composition may include modified
nucleomonomers as discussed above.
The invention also relates to a method to inhibit expression of a
target gene in an invertebrate organism. Such methods include
providing an invertebrate organism containing a target cell that
contains the target gene, in which the target cell is susceptible
to RNA interference and the target gene is expressed in the target
cell. Such methods further include contacting the invertebrate
organism with an RNA composition of the invention. For example, the
RNA may be a double-stranded molecule with a first strand
consisting essentially of a ribonucleotide sequence that
corresponds to a nucleotide sequence of the target gene and a
second strand consisting essentially of a ribonucleotide sequence
that is complementary to the nucleotide sequence of the target
gene. In such cases, the first and the second ribonucleotide
sequences may be separate complementary strands or joined by a
loop, and they hybridize to each other to form the double-stranded
molecule. Finally, such methods include a step of introducing the
duplex RNA composition into the target cell to thereby inhibiting
expression of the target gene.
In one embodiment, the oligonucleotides of the invention can be
used to inhibit gene function in vitro in a method for identifying
the functions of genes. In this manner, the transcription of genes
that are identified, but for which no function has yet been shown,
can be inhibited to thereby determine how the phenotype of a cell
is changed when the gene is not transcribed. Such methods are
useful for the validation of genes as targets for clinical
treatment, e.g., with oligonucleotides or with other therapies.
To determine the effect of a composition of the invention, a
variety of end points can be used. In addition to the assays
described previously herein, for example, nucleic acid probes
(e.g., in the form of arrays) can be used to evaluate transcription
patterns produced by cells. Probes can also be used detect
peptides, proteins, or protein domains, e.g., antibodies can be
used to detect the expression of a particular protein. In yet
another embodiment, the function of a protein (e.g., enzymatic
activity) can be measured. In yet another embodiment, the phenotype
of a cell can be evaluated to determine whether or not a target
protein is expressed. For example, the ability of a composition to
affect a phenotype of a cell that is associated with cancer can be
tested.
In one embodiment, one or more additional agents (e.g., activating
agents, inducing agents, proliferation enhancing agents, tumor
promoters) can be added to the cells.
In another embodiment, the compositions of the invention can be
used to monitor biochemical reactions such as, e.g., interactions
of proteins, nucleic acids, small molecules, or the like, for
example the efficiency or specificity of interactions between
antigens and antibodies; or of receptors (such as purified
receptors or receptors bound to cell membranes) and their ligands,
agonists or antagonists; or of enzymes (such as proteases or
kinases) and their substrates, or increases or decreases in the
amount of substrate converted to a product; as well as many others.
Such biochemical assays can be used to characterize properties of
the probe or target, or as the basis of a screening assay. For
example, to screen samples for the presence of particular proteases
(e.g., proteases involved in blood clotting such as proteases Xa
and VIIa), the samples can be assayed, for example using probes
which are fluorogenic substrates specific for each protease of
interest. If a target protease binds to and cleaves a substrate,
the substrate will fluoresce, usually as a result, e.g., of
cleavage and separation between two energy transfer pairs, and the
signal can be detected. In another example, to screen samples for
the presence of a particular kinase(s) (e.g., a tyrosine kinase),
samples containing one or more kinases of interest can be assayed,
e.g., using probes are peptides which can be selectively
phosphorylated by one of the kinases of interest. Using
art-recognized, routinely determinable conditions, samples can be
incubated with an array of substrates, in an appropriate buffer and
with the necessary cofactors, for an empirically determined period
of time. If necessary, reactions can be stopped, e.g., by washing
and the phosphorylated substrates can be detected by, for example,
incubating them with detectable reagents such as, e.g.,
fluorescein-labeled anti-phosphotyrosine or anti-phosphoserine
antibodies and the signal can be detected.
In another embodiment, the compositions of the invention can be
used to screen for agents which modulate a pattern of gene
expression. Arrays of oligonucleotides can be used, for example, to
identify mRNA species whose pattern of expression from a set of
genes is correlated with a particular physiological state or
developmental stage, or with a disease condition ("correlative"
genes, RNAs, or expression patterns). By the terms "correlate" or
"correlative," it is meant that the synthesis pattern of RNA is
associated with the physiological condition of a cell, but not
necessarily that the expression of a given RNA is responsible for
or is causative of a particular physiological state. For example, a
small subset of mRNAs can be identified which are modulated (e.g.,
upregulated or downregulated) in cells which serve as a model for a
particular disease state. This altered pattern of expression as
compared to that in a normal cell, which does not exhibit a
pathological phenotype, can serve as a indicator of the disease
state ("indicator" or "correlative" genes, RNAs, or expression
patterns).
Compositions which modulate the chosen indicator expression pattern
(e.g., compared to control compositions comprising, for example
oligonucleotides which comprise a nucleotide sequence which is the
reverse of the oligonucleotide, or which contains mismatch bases)
can indicate that a particular target gene is a potential target
for therapeutic intervention. Moreover, such compositions may be
useful as therapeutic agents to modulate expression patters of
cells in an in vitro expression system or in in vivo therapy. As
used herein, "modulate" means to cause to increase or decrease the
amount or activity of a molecule or the like which is involved in a
measurable reaction. In one embodiment, a series of cells (e.g.,
from a disease model) can be contacted with a series of agents
(e.g., for a period of time ranging from about 10 minutes to about
48 hours or more) and, using routine, art-recognized methods (e.g.,
commercially available kits), total RNA or mRNA extracts can be
made. If it is desired to amplify the amount of RNA, standard
procedures such as RT-PCR amplification can be used (see, e.g.,
Innis et al eds., (1996) PCR Protocols: A Guide to Methods in
Amplification, Academic Press, New York). The extracts (or
amplified products from them) can be allowed to contact (e.g.,
incubate with) probes for appropriate indicator RNAs, and those
agents which are associated with a change in the indicator
expression pattern can be identified.
Similarly, agents can be identified which modulate expression
patterns associated with particular physiological states or
developmental stages. Such agents can be man-made or
naturally-occurring substances, including environmental factors
such as substances involved in embryonic development or in
regulating physiological reactions.
In one embodiment, the methods described herein can be performed in
a "high throughput" manner, in which a large number of target genes
(e.g., as many as about 1000 or more, depending on the particular
format used) are assayed rapidly and concurrently. Further, many
assay formats (e.g., plates or surfaces) can be processed at one
time. For example, because the oligonucleotides of the invention do
not need to be tested individually before incorporating them into a
composition, they can be readily synthesized and large numbers of
target genes can be tested at one time. For example, a large number
of samples, each comprising a biological sample containing a target
nucleic acid molecule (e.g., a cell) and a composition of the
invention can be added to separate regions of an assay format and
assays can be performed on each of the samples.
Administration of Oligonucleotide Compositions
The optimal course of administration or delivery of the
oligonucleotides may vary depending upon the desired result and/or
on the subject to be treated. As used herein "administration"
refers to contacting cells with oligonucleotides and can be
performed in vitro or in vivo. The dosage of oligonucleotides may
be adjusted to optimally reduce expression of a protein translated
from a target nucleic acid molecule, e.g., as measured by a readout
of RNA stability or by a therapeutic response, without undue
experimentation.
For example, expression of the protein encoded by the nucleic acid
target can be measured to determine whether or not the dosage
regimen needs to be adjusted accordingly. In addition, an increase
or decrease in RNA or protein levels in a cell or produced by a
cell can be measured using any art recognized technique. By
determining whether transcription has been decreased, the
effectiveness of the oligonucleotide in inducing the cleavage of a
target RNA can be determined.
Any of the above-described oligonucleotide compositions can be used
alone or in conjunction with a pharmaceutically acceptable carrier.
As used herein, "pharmaceutically acceptable carrier" includes
appropriate solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like. The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, it can be used in the therapeutic compositions.
Supplementary active ingredients can also be incorporated into the
compositions.
Oligonucleotides may be incorporated into liposomes or liposomes
modified with polyethylene glycol or admixed with cationic lipids
for parenteral administration. Incorporation of additional
substances into the liposome, for example, antibodies reactive
against membrane proteins found on specific target cells, can help
target the oligonucleotides to specific cell types.
Moreover, the present invention provides for administering the
subject oligonucleotides with an osmotic pump providing continuous
infusion of such oligonucleotides, for example, as described in
Rataiczak et al. (1992 Proc. Natl. Acad. Sci. USA 89:11823-11827).
Such osmotic pumps are commercially available, e.g., from Alzet
Inc. (Palo Alto, Calif.). Topical administration and parenteral
administration in a cationic lipid carrier are preferred.
With respect to in vivo applications, the formulations of the
present invention can be administered to a patient in a variety of
forms adapted to the chosen route of administration, e.g.,
parenterally, orally, or intraperitoneally. Parenteral
administration, which is preferred, includes administration by the
following routes: intravenous; intramuscular; interstitially;
intraarterially; subcutaneous; intra ocular; intrasynovial; trans
epithelial, including transdermal; pulmonary via inhalation;
ophthalmic; sublingual and buccal; topically, including ophthalmic;
dermal; ocular; rectal; and nasal inhalation via insufflation.
Pharmaceutical preparations for parenteral administration include
aqueous solutions of the active compounds in water-soluble or
water-dispersible form. In addition, suspensions of the active
compounds as appropriate oily injection suspensions may be
administered. Suitable lipophilic solvents or vehicles include
fatty oils, for example, sesame oil, or synthetic fatty acid
esters, for example, ethyl oleate or triglycerides. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension include, for example, sodium
carboxymethyl cellulose, sorbitol, or dextran, optionally, the
suspension may also contain stabilizers. The oligonucleotides of
the invention can be formulated in liquid solutions, preferably in
physiologically compatible buffers such as Hank's solution or
Ringer's solution. In addition, the oligonucleotides may be
formulated in solid form and redissolved or suspended immediately
prior to use. Lyophilized forms are also included in the
invention.
Pharmaceutical preparations for topical administration include
transdermal patches, ointments, lotions, creams, gels, drops,
sprays, suppositories, liquids and powders. In addition,
conventional pharmaceutical carriers, aqueous, powder or oily
bases, or thickeners may be used in pharmaceutical preparations for
topical administration.
Pharmaceutical preparations for oral administration include powders
or granules, suspensions or solutions in water or non-aqueous
media, capsules, sachets or tablets. In addition, thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids, or
binders may be used in pharmaceutical preparations for oral
administration.
For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are known in the art, and include, for
example, for transmucosal administration bile salts and fusidic
acid derivatives, and detergents. Transmucosal administration may
be through nasal sprays or using suppositories. For oral
administration, the oligonucleotides are formulated into
conventional oral administration forms such as capsules, tablets,
and tonics. For topical administration, the oligonucleotides of the
invention are formulated into ointments, salves, gels, or creams as
known in the art.
Drug delivery vehicles can be chosen e.g., for in vitro, for
systemic, or for topical administration. These vehicles can be
designed to serve as a slow release reservoir or to deliver their
contents directly to the target cell. An advantage of using some
direct delivery drug vehicles is that multiple molecules are
delivered per uptake. Such vehicles have been shown to increase the
circulation half-life of drugs that would otherwise be rapidly
cleared from the blood stream. Some examples of such specialized
drug delivery vehicles which fall into this category are liposomes,
hydrogels, cyclodextrins, biodegradable nanocapsules, and
bioadhesive microspheres.
The described oligonucleotides may be administered systemically to
a subject. Systemic absorption refers to the entry of drugs into
the blood stream followed by distribution throughout the entire
body. Administration routes which lead to systemic absorption
include: intravenous, subcutaneous, intraperitoneal, and
intranasal. Each of these administration routes delivers the
oligonucleotide to accessible diseased cells. Following
subcutaneous administration, the therapeutic agent drains into
local lymph nodes and proceeds through the lymphatic network into
the circulation. The rate of entry into the circulation has been
shown to be a function of molecular weight or size. The use of a
liposome or other drug carrier localizes the oligonucleotide at the
lymph node. The oligonucleotide can be modified to diffuse into the
cell, or the liposome can directly participate in the delivery of
either the unmodified or modified oligonucleotide into the
cell.
The chosen method of delivery will result in entry into cells.
Preferred delivery methods include liposomes (10-400 nm),
hydrogels, controlled-release polymers, and other pharmaceutically
applicable vehicles, and microinjection or electroporation (for ex
vivo treatments).
The pharmaceutical preparations of the present invention may be
prepared and formulated as emulsions. Emulsions are usually
heterogeneous systems of one liquid dispersed in another in the
form of droplets usually exceeding 0.1 .mu.m in diameter.
The emulsions of the present invention may contain excipients such
as emulsifiers, stabilizers, dyes, fats, oils, waxes, fatty acids,
fatty alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives, and anti-oxidants may also be present in emulsions
as needed. These excipients may be present as a solution in either
the aqueous phase, oily phase or itself as a separate phase.
Examples of naturally occurring emulsifiers that may be used in
emulsion formulations of the present invention include lanolin,
beeswax, phosphatides, lecithin and acacia. Finely divided solids
have also been used as good emulsifiers especially in combination
with surfactants and in viscous preparations. Examples of finely
divided solids that may be used as emulsifiers include polar
inorganic solids, such as heavy metal hydroxides, nonswelling clays
such as bentonite, attapulgite, hectorite, kaolin, montmorillonite,
colloidal aluminum silicate and colloidal magnesium aluminum
silicate, pigments and nonpolar solids such as carbon or glyceryl
tristearate.
Examples of preservatives that may be included in the emulsion
formulations include methyl paraben, propyl paraben, quaternary
ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic
acid, and boric acid. Examples of antioxidants that may be included
in the emulsion formulations include free radical scavengers such
as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated
hydroxytoluene, or reducing agents such as ascorbic acid and sodium
metabisulfite, and antioxidant synergists such as citric acid,
tartaric acid, and lecithin.
In one embodiment, the compositions of oligonucleotides are
formulated as microemulsions. A microemulsion is a system of water,
oil and amphiphile which is a single optically isotropic and
thermodynamically stable liquid solution. Typically microemulsions
are prepared by first dispersing an oil in an aqueous surfactant
solution and then adding a sufficient amount of a 4th component,
generally an intermediate chain-length alcohol to form a
transparent system.
Surfactants that may be used in the preparation of microemulsions
include, but are not limited to, ionic surfactants, non-ionic
surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol
fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol
monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol
pentaoleate (PO500), decaglycerol monocaprate (MCA750),
decaglycerol monooleate (MO750), decaglycerol sequioleate (S0750),
decaglycerol decaoleate (DA0750), alone or in combination with
cosurfactants. The cosurfactant, usually a short-chain alcohol such
as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules.
Microemulsions may, however, be prepared without the use of
cosurfactants and alcohol-free self-emulsifying microemulsion
systems are known in the art. The aqueous phase may typically be,
but is not limited to, water, an aqueous solution of the drug,
glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and
derivatives of ethylene glycol. The oil phase may include, but is
not limited to, materials such as Captex 300, Captex 355, Capmul
MCM, fatty acid esters, medium chain (C.sub.8-C.sub.12) mono, di,
and tri-glycerides, polyoxyethylated glyceryl fatty acid esters,
fatty alcohols, polyglycolized glycerides, saturated polyglycolized
C.sub.8-C.sub.10 glycerides, vegetable oils and silicone oil.
Microemulsions are particularly of interest from the standpoint of
drug solubilization and the enhanced absorption of drugs. Lipid
based microemulsions (both oil/water and water/oil) have been
proposed to enhance the oral bioavailability of drugs.
Microemulsions offer improved drug solubilization, protection of
drug from enzymatic hydrolysis, possible enhancement of drug
absorption due to surfactant-induced alterations in membrane
fluidity and permeability, ease of preparation, ease of oral
administration over solid dosage forms, improved clinical potency,
and decreased toxicity (Constantinides et al., Pharmaceutical
Research, 1994, 11:1385; Ho et al., J. Pharm. Sci., 1996,
85:138-143). Microemulsions have also been effective in the
transdermal delivery of active components in both cosmetic and
pharmaceutical applications. It is expected that the microemulsion
compositions and formulations of the present invention will
facilitate the increased systemic absorption of oligonucleotides
from the gastrointestinal tract, as well as improve the local
cellular uptake of oligonucleotides within the gastrointestinal
tract, vagina, buccal cavity and other areas of administration.
In an embodiment, the present invention employs various penetration
enhancers to affect the efficient delivery of nucleic acids,
particularly oligonucleotides, to the skin of animals. Even
non-lipophilic drugs may cross cell membranes if the membrane to be
crossed is treated with a penetration enhancer. In addition to
increasing the diffusion of non-lipophilic drugs across cell
membranes, penetration enhancers also act to enhance the
permeability of lipophilic drugs.
Five categories of penetration enhancers that may be used in the
present invention include: surfactants, fatty acids, bile salts,
chelating agents, and non-chelating non-surfactants. Other agents
may be utilized to enhance the penetration of the administered
oligonucleotides include: glycols such as ethylene glycol and
propylene glycol, pyrrols such as 2-15 pyrrol, azones, and terpenes
such as limonene, and menthone.
The oligonucleotides, especially in lipid formulations, can also be
administered by coating a medical device, for example, a catheter,
such as an angioplasty balloon catheter, with a cationic lipid
formulation. Coating may be achieved, for example, by dipping the
medical device into a lipid formulation or a mixture of a lipid
formulation and a suitable solvent, for example, an aqueous-based
buffer, an aqueous solvent, ethanol, methylene chloride, chloroform
and the like. An amount of the formulation will naturally adhere to
the surface of the device which is subsequently administered to a
patient, as appropriate. Alternatively, a lyophilized mixture of a
lipid formulation may be specifically bound to the surface of the
device. Such binding techniques are described, for example, in K.
Ishihara et al., Journal of Biomedical Materials Research, Vol. 27,
pp. 1309-1314 (1993), the disclosures of which are incorporated
herein by reference in their entirety.
The useful dosage to be administered and the particular mode of
administration will vary depending upon such factors as the cell
type, or for in vivo use, the age, weight and the particular animal
and region thereof to be treated, the particular oligonucleotide
and delivery method used, the therapeutic or diagnostic use
contemplated, and the form of the formulation, for example,
suspension, emulsion, micelle or liposome, as will be readily
apparent to those skilled in the art. Typically, dosage is
administered at lower levels and increased until the desired effect
is achieved. When lipids are used to deliver the oligonucleotides,
the amount of lipid compound that is administered can vary and
generally depends upon the amount of oligonucleotide agent being
administered. For example, the weight ratio of lipid compound to
oligonucleotide agent is preferably from about 1:1 to about 15:1,
with a weight ratio of about 5:1 to about 10:1 being more
preferred. Generally, the amount of cationic lipid compound which
is administered will vary from between about 0.1 milligram (mg) to
about 1 gram (g). By way of general guidance, typically between
about 0.1 mg and about 10 mg of the particular oligonucleotide
agent, and about 1 mg to about 100 mg of the lipid compositions,
each per kilogram of patient body weight, is administered, although
higher and lower amounts can be used.
The agents of the invention are administered to subjects or
contacted with cells in a biologically compatible form suitable for
pharmaceutical administration. By "biologically compatible form
suitable for administration" is meant that the oligonucleotide is
administered in a form in which any toxic effects are outweighed by
the therapeutic effects of the oligonucleotide. In one embodiment,
oligonucleotides can be administered to subjects. Examples of
subjects include mammals, e.g., humans and other primates; cows,
pigs, horses, and farming (agricultural) animals; dogs, cats, and
other domesticated pets; mice, rats, and transgenic non-human
animals.
Administration of an active amount of an oligonucleotide of the
present invention is defined as an amount effective, at dosages and
for periods of time necessary to achieve the desired result. For
example, an active amount of an oligonucleotide may vary according
to factors such as the type of cell, the oligonucleotide used, and
for in vivo uses the disease state, age, sex, and weight of the
individual, and the ability of the oligonucleotide to elicit a
desired response in the individual. Establishment of therapeutic
levels of oligonucleotides within the cell is dependent upon the
rates of uptake and efflux or degradation. Decreasing the degree of
degradation prolongs the intracellular half-life of the
oligonucleotide. Thus, chemically-modified oligonucleotides, e.g.,
with modification of the phosphate backbone, may require different
dosing.
The exact dosage of an oligonucleotide and number of doses
administered will depend upon the data generated experimentally and
in clinical trials. Several factors such as the desired effect, the
delivery vehicle, disease indication, and the route of
administration, will affect the dosage. Dosages can be readily
determined by one of ordinary skill in the art and formulated into
the subject pharmaceutical compositions. Preferably, the duration
of treatment will extend at least through the course of the disease
symptoms.
Dosage regima may be adjusted to provide the optimum therapeutic
response. For example, the oligonucleotide may be repeatedly
administered, e.g., several doses may be administered daily or the
dose may be proportionally reduced as indicated by the exigencies
of the therapeutic situation. One of ordinary skill in the art will
readily be able to determine appropriate doses and schedules of
administration of the subject oligonucleotides, whether the
oligonucleotides are to be administered to cells or to
subjects.
Treatment of Diseases or Disorders
By inhibiting the expression of a gene, the oligonucleotide
compositions of the present invention can be used to treat any
disease involving the expression of a protein. Examples of diseases
that can be treated by oligonucleotide compositions include:
cancer, retinopathies, autoimmune diseases, inflammatory diseases
(i.e., ICAM-1 related disorders, Psoriasis, Ulcerative Colitus,
Crohn's disease), viral diseases (i.e., HIV, Hepatitis C), and
cardiovascular diseases.
In one embodiment, in vitro treatment of cells with
oligonucleotides can be used for ex vivo therapy of cells removed
from a subject (e.g., for treatment of leukemia or viral infection)
or for treatment of cells which did not originate in the subject,
but are to be administered to the subject (e.g., to eliminate
transplantation antigen expression on cells to be transplanted into
a subject). In addition, in vitro treatment of cells can be used in
non-therapeutic settings, e.g., to evaluate gene function, to study
gene regulation and protein synthesis or to evaluate improvements
made to oligonucleotides designed to modulate gene expression or
protein synthesis. In vivo treatment of cells can be useful in
certain clinical settings where it is desirable to inhibit the
expression of a protein. There are numerous medical conditions for
which antisense therapy is reported to be suitable (see, e.g., U.S.
Pat. No. 5,830,653) as well as respiratory syncytial virus
infection (WO 95/22,553) influenza virus (WO 94/23,028), and
malignancies (WO 94/08,003). Other examples of clinical uses of
antisense sequences are reviewed, e.g., in Glaser. 1996. Genetic
Engineering News 16:1. Exemplary targets for cleavage by
oligonucleotides include, e.g., protein kinase Ca, ICAM-1, c-raf
kinase, p53, c-myb, and the bcr/abl fusion gene found in chronic
myelogenous leukemia.
The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of cell biology, cell culture,
molecular biology, microbiology, recombinant DNA, and immunology,
which are within the skill of the art. Such techniques are
explained fully in the literature. See, for example, Molecular
Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, J. et al.
(Cold Spring Harbor Laboratory Press (1989)); Short Protocols in
Molecular Biology, 3rd Ed., ed. by Ausubel, F. et al. (Wiley, NY
(1995)); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985);
Oligonucleotide Synthesis (M. J. Gait ed. (1984)); Mullis et al.
U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames
& S. J. Higgins eds. (1984)); the treatise, Methods In
Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In
Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,
London (1987)); Handbook Of Experimental Immunology, Volumes I-IV
(D. M. Weir and C. C. Blackwell, eds. (1986)); and Miller, J.
Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold
Spring Harbor, N.Y. (1972)).
Business Methods
The present invention also provides a system and method of
providing company products to a party outside of the company, for
example, a system and method for providing a customer or a product
distributor a product of the company such as a kit containing a
double stranded nucleic acid molecule which is capable of
inhibiting expression of a gene and/or instructions for inhibiting
gene expression. FIG. 7 provides a schematic diagram of a product
management system. In practice, the blocks in FIG. 7 can represent
an intra-company organization, which can include departments in a
single building or in different buildings, a computer program or
suite of programs maintained by one or more computers, a group of
employees, a computer I/O device such as a printer or fax machine,
a third party entity or company that is otherwise unaffiliated with
the company, or the like.
The product management system as shown in FIG. 7 is exemplified by
company 100, which receives input in the form of an order from a
party outside of the company, e.g., distributor 150 or customer
140, to order department 126, or in the form of materials and parts
130 from a party outside of the company; and provides output in the
form of a product delivered from shipping department 119 to
distributor 150 or customer 140. Company 100 system is organized to
optimize receipt of orders and delivery of a products to a party
outside of the company in a cost efficient manner, particularly
instructions or a kit of the present invention, and to obtain
payment for such product from the party.
With respect to methods of the present invention, the term
"materials and parts" refers to items that are used to make a
device, other component, or product, which generally is a device,
other component, or product that company sells to a party outside
of the company. As such, materials and parts include, for example,
nucleotides, single stranded or double stranded nucleic acid
molecules, host cells, enzymes (e.g., polymerases), amino acids,
culture media, buffers, paper, ink, reaction vessels, etc. In
comparison, the term "devices", "other components", and "products"
refer to items sold by the company. Devices are exemplified by
nucleic acid molecules that are to be sold by the company, for
example, single stranded or double stranded nucleic acid molecules
which may or may not contain one or more chemical modifications in
one or both strands. Other components are exemplified by
instructions, including instructions for determining a ratio of
nucleic acid molecules to be combined with cells for optimal
inhibition of gene expression according to a method of the
invention. Other components also can be items that may be included
in a kit, e.g., a kit product containing, for example, single
stranded or double stranded nucleic acid molecules or cells of one
or more type (e.g., 293 cells, HUVEC cells, etc). As such, it will
be recognized that an item useful as materials and parts as defined
herein further can be considered an "other component", which can be
sold by the company. The term "products" refers to devices, other
components, or combinations thereof, including combinations with
additional materials and parts, that are sold or desired to be sold
or otherwise provided by a company to one or more parties outside
of the company. Products are exemplified herein by kits, which can
contain instructions according to the present invention, and single
stranded or double stranded nucleic acid molecules, or combinations
thereof.
Referring to FIG. 7, company 100 includes manufacturing 110 and
administration 120. Devices 112 and other components 114 are
produced in manufacturing 110, and can be stored separately therein
such as in device storage 113 and other component storage 115,
respectively, or can be further assembled and stored in product
storage 117. Materials and parts 130 can be provided to company 100
from an outside source and/or materials and parts 114 can be
prepared in company, and used to produce devices 112 and other
components 116, which, in turn, can be assembled and sold as a
product. Manufacturing 110 also includes shipping department 119,
which, upon receiving input as to an order, can obtain products to
be shipped from product storage 117 and forward the product to a
party outside the company.
For purposes of the present invention, product storage 117 can
store instructions, for example, for determining transfection
conditions which are suitable for use with a particular cell type
or how to design a double stranded nucleic acid molecule which will
function for inhibiting gene expression, as well as combinations of
such instructions and/or kits. Upon receiving input from order
department 126, for example, that customer 140 has ordered such a
kit and instructions, shipping department 119 can obtain from
product storage 117 such kit for shipping, and can further obtain
such instructions in a written form to include with the kit, and
ship the kit and instructions to customer 140 (and providing input
to billing department 124 that the product was shipped; or shipping
department 119 can obtain from product storage 117 the kit for
shipping, and can further provide the instructions to customer 140
in an electronic form, by accessing a database in company 100 that
contains the instructions, and transmitting the instructions to
customer 140 via the internet (not shown).
As further exemplified in FIG. 7, administration 120 includes order
department 126, which receives input in the form of an order for a
product from customer 140 or distributor 150. Order department 126
then provides output in the form of instructions to shipping
department 119 to fill the order, i.e., to forward products as
requested to customer 140 or distributor 150. Shipping department
119, in addition to filling the order, further provides input to
billing department 124 in the form of confirmation of the products
that have been shipped. Billing department 124 then can provide
output in the form of a bill to customer 140 or distributor 150 as
appropriate, and can further receive input that the bill has been
paid, or, if no such input is received, can further provide output
to customer 140 or distributor 150 that such payment may be
delinquent. Additional optional component of company 100 include
customer service department 122, which can receive input from
customer 140 and can provide output in the form of feedback or
information to customer 140. Furthermore, although not shown in
FIG. 7, customer service 122 can receive input or provide output to
any other component of company. For example, customer service
department 122 can receive input from customer 140 indicating that
an ordered product was not received, wherein customer service
department 122 can provide output to shipping department 119 and/or
order department 126 and/or billing department 124 regarding the
missing product, thus providing a means to assure customer 140
satisfaction. Customer service department 122 also can receive
input from customer 140 in the form of requested technical
information, for example, for confirming that instructions of the
invention can be applied to the particular need of customer 140,
and can provide output to customer 140 in the form of a response to
the requested technical information.
As such, the components of company 100 are suitably configured to
communicate with each other to facilitate the transfer of materials
and parts, devices, other components, products, and information
within company 100, and company 100 is further suitably configured
to receive input from or provide output to an outside party. For
example, a physical path can be utilized to transfer products from
product storage 117 to shipping department 119 upon receiving
suitable input from order department 126. Order department 126, in
comparison, can be linked electronically with other components
within company 100, for example, by a communication network such as
an intranet, and can be further configured to receive input, for
example, from customer 140 by a telephone network, by mail or other
carrier service, or via the internet. For electronic input and/or
output, a direct electronic link such as a T1 line or a direct
wireless connection also can be established, particularly within
company 100 and, if desired, with distributor 150 or materials or
parts 130 provider, or the like.
Although not illustrated, company 100 system one or more data
collection systems, including, for example, a customer data
collection system, which can be realized as a personal computer, a
computer network, a personal digital assistant (PDA), an audio
recording medium, a document in which written entries are made, any
suitable device capable of receiving data, or any combination of
the foregoing. Data collection systems can be used to gather data
associated with a customer 140 or distributor 150, including, for
example, a customer's shipping address and billing address, as well
as more specific information such as the customer's ordering
history and payment history, such data being useful, for example,
to determine that a customer has made sufficient purchases to
qualify for a discount on one or more future purchases.
Company 100 can utilize a number of software applications to
provide components of company 100 with information or to provide a
party outside of company access to one or more components of
company 100, for example, access to order department 126 or
customer service department 122. Such software applications can
comprise a communication network such as the Internet, a local area
network, or an intranet. For example, in an internet-based
application, customer 140 can access a suitable web site and/or a
web server that cooperates with order department 126 such that
customer 140 can provide input in the form of an order to order
department 126. In response, order department 126 can communicate
with customer 140 to confirm that the order has been received, and
can further communicate with shipping department 119, providing
input that products such as a kit of the invention, which contains,
for example, a double-stranded nucleic acid molecule and
instructions for use, should be shipped to customer 140. In this
manner, the business of company 100 can proceed in an efficient
manner.
In a networked arrangement, billing department 124 and shipping
department 119, for example, can communicate with one another by
way of respective computer systems. As used herein, the term
"computer system" refers to general purpose computer systems such
as network servers, laptop systems, desktop systems, handheld
systems, personal digital assistants, computing kiosks, and the
like. Similarly, in accordance with known techniques, distributor
150 can access a web site maintained by company 100 after
establishing an online connection to the network, particularly to
order department 126, and can provide input in the form of an
order. If desired, a hard copy of an order placed with order
department 126 can be printed from the web browser application
resident at distributor 150.
The various software modules associated with the implementation of
the present invention can be suitably loaded into the computer
systems resident at company 100 and any party outside of company
100 as desired, or the software code can be stored on a
computer-readable medium such as a floppy disk, magnetic tape, or
an optical disk. In an online implementation, a server and web site
maintained by company 100 can be configured to provide software
downloads to remote users such as distributor 150, materials and
parts 130, and the like. When implemented in software, the
techniques of the present invention are carried out by code
segments and instructions associated with the various process tasks
described herein.
Accordingly, the present invention further includes methods for
providing various aspects of a product (e.g., a kit and/or
instructions of the invention), as well as information regarding
various aspects of the invention, to parties such as the parties
shown as customer 140 and distributor 150 in FIG. 7. Thus, methods
for selling devices, products and methods of the invention to such
parties are provided, as are methods related to those sales,
including customer support, billing, product inventory management
within the company, etc. Examples of such methods are shown in FIG.
7, including, for example, wherein materials and parts 130 can be
acquired from a source outside of company 100 (e.g., a supplier)
and used to prepare devices (e.g., double-stranded nucleic acid
molecules) used in preparing a composition or practicing a method
of the invention, for example, kits, which can be maintained as an
inventory in product storage 117. It should be recognized that
devices 112 can be sold directly to a customer and/or distributor
(not shown), or can be combined with one or more other components
116, and sold to a customer and/or distributor as the combined
product. The other components 116 can be obtained from a source
outside of company 100 (materials and parts 130) or can be prepared
within company 100 (materials and parts 114). As such, the term
"product" is used generally herein to refer an item sent to a party
outside of the company (a customer, a distributor, etc.) and
includes items such as devices 112, which can be sent to a party
alone or as a component of a kit or the like.
At the appropriate time, the product is removed from product
storage 117, for example, by shipping department 119, and sent to a
requesting party such as customer 140 or distributor 150.
Typically, such shipping occurs in response to the party placing an
order, which is then forwarded the within the organization as
exemplified in FIG. 7, and results in the ordered product being
sent to the party. Data regarding shipment of the product to the
party is transmitted further within the organization, for example,
from shipping department 119 to billing department 124, which, in
turn, can transmit a bill to the party, either with the product, or
at a time after the product has been sent. Further, a bill can be
sent in instances where the party has not paid for the product
shipped within a certain period of time (e.g., within 30 days,
within 45 days, within 60 days, within 90 days, within 120 days,
within from 30 days to 120 days, within from 45 days to 120 days,
within from 60 days to 120 days, within from 90 days to 120 days,
within from 30 days to 90 days, within from 30 days to 60 days,
within from 30 days to 45 days, within from 60 days to 90 days,
etc.). Typically, billing department 124 also is responsible for
processing payment(s) made by the party. It will be recognized that
variations from the exemplified method can be utilized; for
example, customer service department 122 can receive an order from
the party, and transmit the order to shipping department 119 (not
shown), thus serving the functions exemplified in FIG. 7 by order
department 126 and the customer service department 122.
Methods of the invention also include providing technical service
to parties using a product, particularly a kit of the invention.
While such a function can be performed by individuals involved in
product research and development, inquiries related to technical
service generally are handled, routed, and/or directed by an
administrative department of the organization (e.g., customer
service department 122). Often communications related to technical
service (e.g., solving problems related to use of the product or
individual components of the product) require a two way exchange of
information, as exemplified by arrows indicating pathways of
communication between customer 150 and customer service department
122.
As mentioned above, any number of variations of the process
exemplified in FIG. 7 are possible and within the scope of the
invention. Accordingly, the invention includes methods (e.g.,
business methods) that involve (1) the production of products
(e.g., double-stranded nucleic acid molecules, transfection
reagents, kits that contain instructions for performing methods of
the invention, etc.); (2) receiving orders for these products; (3)
sending the products to parties placing such orders; (4) sending
bills to parties obliged to pay for products sent to such; and/or
(5) receiving payment for products sent to parties. For example,
methods are provided that comprise two or more of the following
steps: (a) obtaining parts, materials, and/or components from a
supplier; (b) preparing one or more first products (e.g., one or
more double-stranded nucleic acid molecules); (c) storing the one
or more first products of step (b); (d) combining the one or more
first products of step (b) with one or more other components to
form one or more second products (e.g., a kit); (e) storing the one
or more first products of step (b) or one or more second products
of step (d); (f) obtaining an order a first product of step (b) or
a second product of step (d); (g) shipping either the first product
of step (b) or the second product of step (d) to the party that
placed the order of step (f); (h) tracking data regarding to the
amount of money owed by the party to which the product is shipped
in step (g); (i) sending a bill to the party to which the product
is shipped in step (g); (j) obtaining payment for the product
shipped in step (g) (generally, but not necessarily, the payment is
made by the party to which the product was shipped in step (g); and
(k) exchanging technical information between the organization and a
party in possession of a product shipped in step (d) (typically,
the party to which the product was shipped in step (g)).
The present invention also provides a system and method for
providing information as to availability of a product (e.g., a
device product, a kit product, and the like) to parties having
potential interest in the availability of the kit product. Such a
method of the invention, which encompasses a method of advertising
to the general or a specified public, the availability of the
product, particularly a product comprising instructions and/or a
kit of the present invention, can be performed, for example, by
transmitting product description data to an output source, for
example, an advertiser; further transmitting to the output source
instructions to publish the product information data in media
accessible to the potential interested parties; and detecting
publication of the data in the media, thereby providing information
as to availability of the product to parties having potential
interest in the availability of the product.
Accordingly, the present invention provides methods for advertising
and/or marketing devices, products, and/or methods of the
invention, such methods providing the advantage of inducing and/or
increasing the sales of such devices, products, and/or methods. For
example, advertising and/or marketing methods of the invention
include those in which technical specifications and/or descriptions
of devices and/or products; methods of using the devices and/or
products; and/or instructions for practicing the methods and/or
using the devices and/or products are presented to potential
interested parties, particularly potential purchasers of the
product such as customers, distributors, and the like. In
particular embodiments, the advertising and/or marketing methods
involve presenting such information in a tangible form or in an
intangible to the potential interested parties. As disclosed herein
and well known in the art, the term "intangible form" means a form
that cannot be physically handled and includes, for example,
electronic media (e.g., e-mail, internet web pages, etc.),
broadcasts (e.g., television, radio, etc.), and direct contacts
(e.g., telephone calls between individuals, between automated
machines and individuals, between machines, etc.); whereas the term
"tangible form" means a form that can be physically handled.
FIG. 8 provides a schematic diagram of an information providing
management system as encompassed within the present invention. In
practice, the blocks in FIG. 8 can represent an intra-company
organization, which can include departments in a single building or
in different buildings, a computer program or suite of programs
maintained by one or more computers, a group of employees, a
computer I/O device such as a printer or fax machine, a third party
entity or company that is otherwise unaffiliated with the company,
or the like.
The information providing management system as shown in FIG. 8 is
exemplified by company 200, which makes, purchases, or otherwise
makes available devices and methods 210 that alone, or in
combination, provide products 220, for example, instructions,
devices and/or kits of the present invention, that company 200
wishes to sell to interested parties. To this end, product
descriptions 230 are made, providing information that would lead
potential users to believe that products 220 can be useful to user.
In order to effect transfer of product descriptions 230 to the
potential users, product descriptions 230 is provided to
advertising agency 240, which can be an entity separate from
company 200, or to advertising department 260, which can be an
entity related to company 200, for example, a subsidiary. Based on
the product descriptions 230, advertisement 250 is generated and is
provided to media accessible to potential purchasers of products
260, whom may then contact company 200 to purchase products
220.
By way of example, product descriptions 230 can be in a tangible
form such as written descriptions, which can be delivered (e.g.,
mailed, couriered, etc) to advertising agency 240 and/or
advertising department 250, or can be in an intangible form such as
entered into and stored in a database (e.g., on a computer, in an
electronic media, etc.) and transmitted to advertising agency 240
and/or advertising department 250 over a telephone line, T1 line,
wireless network, or the like. Similarly, advertisement 250 can be
a tangible or intangible form such that it conveniently and
effectively can be provided to potential parties of interest (e.g.,
potential purchasers of product 260). For example, advertisement
250 can be provided in printed form as flyers (e.g., at a meeting
or other congregation of potential interested parties) or as
printed pages (or portions thereof) in magazines known to be read
by the potential interested parties (e.g., trade magazines,
journals, newspapers, etc.). In addition, or alternatively,
advertisement 250 can be provided in the form of directed mailing
of computer media containing the advertisement (e.g., CDs, DVDs,
floppy discs, etc.) or of e mail (i.e., mail or e-mail that is sent
only to selected parties, for example, parties known to members of
an organization that includes or is likely to include potential
users of products 220); of web pages (e.g., on a website provided
by company 200, or having links to the company 200 website); or of
pop-up or pop-under ads on web pages known to be visited by
potential purchaser of products 260, and the like. Potential
purchasers of products 260, upon being apprised of the availability
of the products 220, for example, the kits of the present
invention, then can contact company 200 and, if so desired, can
order said products 220 for company 200 (see FIG. 7).
Kits and Instructions:
The invention also provides kits. In various aspects, a kit of the
invention may contain one or more (e.g., one, two, three, four,
five, six, seven, etc.) of the following components: (1) one or
more sets of instructions, including, for example, instructions for
performing methods of the invention or for preparing and/or using
compositions of the invention; (2) one or more cells, including,
for example, one or more mammalian cells, for example, cells that
are adapted for growth in a tissue culture medium, (3) one or more
oligonucleotide or double stranded nucleic acid molecule (including
one or more control nucleic acid molecule, as described elsewhere
herein); (4) one or more container containing water (e.g.,
distilled water) or other aqueous or liquid material; (5) one or
more containers containing one or more buffers, which can be
buffers in dry, powder form or reconstituted in a liquid such as
water, including in a concentrated form such as 2.times., 3.times.,
4.times., 5.times., etc.); and/or (6) one or more containers
containing one or more salts (e.g., sodium chloride, potassium
chloride, magnesium chloride, which can be in a dry, powder form or
reconstituted in a liquid such as water).
A kit of the invention can include an instruction set, or the
instructions can be provided independently of a kit. Such
instructions may provide information regarding how to make or use
one or more of the following items: (1) one or more control nucleic
acid molecule (e.g., a nucleic acid molecule which may be used as a
transfection control); (2) one or more double stranded nucleic acid
molecule, as described elsewhere herein (e.g., a double stranded
nucleic acid molecule which is capable of "knocking-down"
expression of a gene where introduced into a eukaryotic cell); (3)
one or more cell lines that contain a gene the expression of which
is to be knocked down (e.g., pre-transfection growth conditions;
transfection protocols; post-transfection growth conditions); (4)
one or more dyes for distinguishing live from dead cells (e.g.,
Dead Red stain or Dead Cell Reagent), and/or (5) one or more sets
of instructions for using kit components.
Instructions can be provided in a kit, for example, written on
paper or in a computer readable form provided with the kit, or can
be made accessible to a user via the internet, for example, on the
world wide web at a URL (uniform resources link; i.e., "address")
specified by the provider of the kit or an agent of the provider.
Such instructions direct a user of the kit or other party of
particular tasks to be performed or of particular ways for
performing a task. In one aspect, the instructions instruct a user
of how to perform methods of the invention. In a specific aspect,
the instructions can, for example, instruct a user of a kit as to
reaction conditions for knocking-down gene expression, including,
for example, buffers, temperature, and/or time periods of
incubations for using nucleic acid molecules described herein.
Instructions of the invention can be in a tangible form, for
example, printed or otherwise imprinted on paper, or in an
intangible form, for example, present on an internet web page at a
defined and accessible URL. Thus, the invention includes
instructions for performing methods of the invention and/or for
preparing compositions of the invention. While the instructions
themselves are one aspect of the invention, the invention also
includes the instructions in tangible form. Thus, the invention
includes computer media (e.g., hard disks, floppy disks, CDs, etc.)
and sheets of paper (e.g., a single sheet of paper, a booklet,
etc.) which contain the instructions.
It will be recognized that a full text of instructions for
performing a method of the invention or, where the instructions are
included with a kit, for using the kit, need not be provided. One
example of a situation in which a kit of the invention, for
example, would not contain such full length instructions is where
the provided directions inform a user of the kits where to obtain
instructions for practicing methods for which the kit can be used.
Thus, instructions for performing methods of the invention can be
obtained from internet web pages, separately sold or distributed
manuals or other product literature, etc. The invention thus
includes kits that direct a kit user to one or more locations where
instructions not directly packaged and/or distributed with the kits
can be found. Such instructions can be in any form including, but
not limited to, electronic or printed forms.
The invention is further illustrated by the following examples,
which should not be construed as further limiting.
EXAMPLES
Example 1
Oligonucleotide Compositions Comprising Chimeric Antisense
Sequences
A gapped antisense oligonucleotide comprising 2'-O-methyl RNA arms
and an unmodified DNA gap was synthesized. A complementary
oligonucleotide was also synthesized using unmodified RNA. A
double-stranded duplex was formed and the composition was found to
inhibit expression of the target gene.
Example 2
Length of Double-Stranded Oligonucleotides and the Presence or
Absence of Overhangs has No Effect on Function
Twenty one and 27-mers were designed to target each of two sites on
the p53 molecule (89-90 site, and 93-94 site). The double-stranded
molecules were designed with or without 3'-deoxy TT overhangs. The
test oligonucleotides were 21-mers with 2 nucleotide 3' deoxy TT
overhangs and without overhangs (blunt ends); and 27-mers with 2
nucleotide 3' deoxy TT overhangs and without overhangs (blunt
ends). Two positive controls were included in the experiment (p53)
and two negative controls were also included (FITC).
A549 cells were transfected with 100 nM of the double-stranded
molecules plus 2 ug/mL LIPOFECTAMINE.TM. 2000. A549 cells were
examined 24 hours post-transfection. FITC-labeled molecules were
taken up well by cells. Both 21-mers (with or without overhangs)
and 27-mers (with or without overhangs) were non-toxic to cells.
FIG. 14A shows the result of an experiment comparing the ability of
different oligonucleotide constructs to inhibit p53 and shows that
length or the presence or absence of a 3' deoxy TT overhang did not
affect the activity of the oligonucleotide. The results in FIG. 14A
show the amount of p53 mRNA normalized to the amount of an
irrelevant message, GAPDH. The level of mRNA was determined using
RT-PCR analysis. The observed percent inhibition of p53 expression
is shown below:
TABLE-US-00006 21-MER 27-MER SITE overhang no overhang overhang no
overhang 93-94 58% 65% 62% 62% 89-90 81% 75% 67% 70%
Similar results were observed for .beta.-3-integrin; both 21-mer
and 27-mer double-stranded molecules were found to inhibit integrin
mRNA. Two double-stranded RNA complexes designed to target the same
site of the .beta.-3-integrin gene were transfected in HMVEC cells.
Both complexes contained a two nucleotide (TT) overhang: one
complex was a 21-mer (with 19 nucleotides complementary to the
target gene) and the other was a 27-mer (with 25 nucleotides
complementary to the target gene). RT-PCR analysis showed that the
two complexes inhibited the target gene to the same extent. HMVEC
cells were transfected using 100 nM oligomer complexed with 2.0
ug/mL of LIPOFECTAMINE.TM. 2000 in media containing serum for 24
hours. Twenty-four hours after transfection, the cells were lysed
and the RNA was isolated for analysis by RT-PCR. No significant
toxicity was observed. The results in FIG. 14B show the amount of
.beta.-3-integrin mRNA normalized to the amount of GAPDH, as
determined by RT-PCR analysis.
Example 3
Activation of the Double-Stranded RNA, Interferon-Inducible Protein
Kinase, PKR
PKR is activated by double-stranded RNA molecules. Active PKR leads
to the inhibition of protein synthesis, activation of
transcription, and a variety of other cellular effects, including
signal transduction, cell differentiation, cell growth inhibition,
apoptosis, and antiviral effects. The effect of p53-targeted
double-stranded RNA molecules on PKR expression was tested. The
level of mRNA was determined using RT-PCR analysis. As shown in
FIG. 15A, no correlation was observed between the length of the
double-stranded oligonucleotide and the level of PKR induction.
Accordingly, long oligonucleotides can be used without activating
PKR, a marker for interferon induction.
As illustrated in FIG. 15B, analysis of relative amounts of PKR
mRNA after the 21- and 27-mer transfection in HMVEC cells showed
approximately a 2 fold increase in PKR mRNA of the siRNA control
sequences over no treatment, and approximately a 2 fold increase of
PKR mRNA of the 27-mer compared to the 21-mer targeted
double-stranded RNA complexes.
Example 4
The Effect of 5' Vs. 3' Modification on the Activity of
Double-Stranded Oligonucleotides
Oligonucleotide duplexes were modified at either the 3' or 5' end
with FITC groups. The modifications were made on either the
antisense strand or the sense strand. 5' or 3' modification of the
sense strand had no effect on the percent inhibition of p53 mRNA.
3' modification of the antisense strand had little affect on
activity, while 5' modification of the antisense strand reduced
activity significantly. 3' modification of both strands also had
little affect on activity, while 3' and 5' modification of both
strands reduced activity. See FIG. 16.
The effect of the size of the group used to modify the 5' end was
tested. The results of this experiment are shown in FIG. 17. The
inclusion of a 5' phosphate group had little effect on activity,
whereas the modification of the antisense strand or both strands
had a greater effect. The inclusion of a propyl group had more of
an effect, with a 5' propyl group on the antisense strand showing a
large reduction in activity; there was also an effect when this
group was added to both strands. Similarly, the inclusion of a FITC
group at the 5' end of the antisense molecule (or to both
molecules) also significantly reduced the activity of the RNA
duplex.
Example 5
Comparison of the Efficacy of 2'-O-Methyl Modified and Unmodified
Double-Stranded RNA Oligonucleotides
A549 cells were transfected with modified or unmodified RNA
duplexes complexed at 100 nM with 2 ug/mL LIPOFECTAMINE.TM. 2000
(Invitrogen) and were transfected for 24 hours. The A549 cells were
plated at 20,000/well in 48 well plates. After 24 hours,
FITC-labeled double-stranded oligonucleotides were visible in A549
cells; the inclusion of a 2'-O-methyl group did not affect uptake.
The Table below shows the results of this experiment.
TABLE-US-00007 2'-O-Methyl Oligonucleotide Duplexes Anti- Anti-
Anti- Anti- sense/Sense sense/Sense sense/Sense sense/Sense
2'-O--Me/2'-O--Me 2'-O--Me/RNA RNA/2'-O--Me RNA/RNA targeted
18639/18640 18639/16194 16193/18640 18876 non-targeted 19039/19040
19039/19044 19043/19040 18850 & 16197/16198 FITC-2'-O--Me/
FITC-2'-O--Me/ FITC-2'-O--Me/ 2'-O--Me/ FITC 2'-O--Me FITC-RNA RNA
FITC-RNA non-targeted 19209 19037/19042 19037/19044 19039/19042
The affect of 2'-O-methyl modifications to one or both strands of a
double-stranded RNA molecule is shown in FIG. 18.
Example 6
Toxicity of p53-Targeted siRNAs in A549 Cells
27-mer siRNAs targeting p53 were not toxic to cells when compared
to standard 21-mer siRNAs having 3' deoxy TT overhangs. In this
experiment, both siRNA constructs inhibited p53 to a similar extent
(83% inhibition for 27-mer vs. 90% inhibition for 21-mer). siRNAs
were designed to target p53 and were constructed as blunt-end
27-mers or as 21-mers with 3' deoxy TT overhangs. A549 cells were
plated at 20,000 cells per well in 48-well plates on the day prior
to transfection. On the day of transfection, cells were
approximately 60-70% confluent. Cells were transfected with 100 nM
siRNAs complexed with 2 ug/mL LIPOFECTAMINE.TM. 2000 for 24 hours.
Following transfection, cells were stained with Dead Red stain to
visualize the extent of cell death. The siRNA sequences used were
as follows:
TABLE-US-00008 21-mer with overhangs targeted (5'-3'):
ACCUCAAAGCUGUUCCGUCTT (SEQ ID NO: 21) GACGGAACAGCUUUGAGGUTT (SEQ ID
NO: 22) Blunt-end 27-mer targeted (5'-3'):
ACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: 23)
GGGACGGAACAGCTTTGAGGTGTGCGT (SEQ ID NO: 24)
Example 7
Toxicity of Blunt-End 27-mer siRNAs Targeting p53 in A549 Cells
The toxicity of targeted blunt-end 27-mer siRNAs targeting p53 was
observed to be not significantly different than a control nucleic
acid or no treatment. siRNAs were designed to target p53 and were
constructed as blunt-end 27-mers. The corresponding control
consisted of chemistry-matched, scrambled sequences with a similar
base-pair composition. A549 cells were plated at 20,000 cells per
well in 48-well plates on the day prior to transfection. On the day
of transfection, cells were approximately 60-70% confluent. Cells
were transfected with 100 nM siRNAs complexed with 2 ug/mL
LIPOFECTAMINE.TM. 2000 for 24 hours. Following transfection, the
cells were stained with Dead Red stain to visualize the extent of
cell death. The siRNA sequences used were as follows:
TABLE-US-00009 Blunt-end 27-mer targeted (5'-3' on top):
ACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: 25)
GGGACGGAACAGCTTTGAGGTGTGCGT (SEQ ID NO: 26) Corresponding control
(5'-3' on top): CCCTGCCTTGTCGAAACTCCACACGCA (SEQ ID NO: 27)
TGCGTGTGGAGTTTCGACAAGGCAGGG (SEQ ID NO: 28)
Example 8
Toxicity of Blunt End 32-mer siRNAs Targeting p53 in A549 Cells
Similarly, blunt-end 32-mer siRNAs targeting p53 were not observed
to be toxic to cells in comparison with a control nucleic acid and
no treatment, as determined by Dead Red staining. siRNAs were
designed to target p53 and were constructed as blunt-end 32-mers.
The corresponding control consisted of chemistry-matched, scrambled
sequences with a similar base-pair composition. A549 cells were
plated at 20,000 cells per well in 48-well plates on the day prior
to transfection. On the day of transfection, cells were
approximately 60-70% confluent. Cells were transfected with 100 nM
siRNAs complexed with 2 ug/mL LIPOFECTAMINE.TM. 2000 for 24 hours.
Following transfection, cells were stained with Dead Red stain to
visualize the extent of cell death. The siRNA sequences used were
as follows:
TABLE-US-00010 Targeted blunt-end 32-mer (5'-3' on top:)
CCCTCACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: 29)
GGGACGGAACAGCTTTGAGGTGTGCGTGAGGG (SEQ ID NO: 30) Corresponding
control (5'-3' on top): CCCTGCCTTGTCGAAACTCCACACGCACTCCC (SEQ ID
NO: 31) GGGAGTGCGTGTGGAGTTTCGACAAGGCAGGG (SEQ ID NO: 32)
Example 9
Inhibition of p53 by 32- and 37-mer Blunt-End siRNAs
FIG. 19 depicts the results of inhibition of p53 by 32- and 37-mer
blunt-end siRNAs in comparison with various control experiments.
siRNAs were designed to target each of two sites (93-93 site) and
(89-90 site) along the coding region of p53. siRNAs were
constructed as blunt-end 32-mers or blunt-end 37-mers. Positive
control siRNAs were 21-mers with 3' deoxy TT overhangs.
Corresponding controls consisted of chemistry-matched, scrambled
sequences with a similar base-pair composition. A549 cells were
plated at 20,000 cells per well in 48-well plates on the day prior
to transfection. On the day of transfection, cells were
approximately 60-70% confluent. Cells were transfected with 100 nM
siRNAs complexed with 2 ug/mL LIPOFECTAMINE.TM. 2000 for 24 hours.
Following transfection, cells were lysed and poly(A) mRNA was
harvested for RT-PCR. Inhibition of p53 expression was determined
by quantitative real-time RT-PCR (TaqMan) analysis. Expression of
p53 was standardized by quantifying GAPDH for each sample. The data
in FIG. 19 represent three separate transfections analyzed in
duplicate and normalized to the internal control (GAPDH). The siRNA
sequences used were as follows (depicted with the 5'-3' strand on
top):
TABLE-US-00011 Targeted 32-mer (89-90 site): (SEQ ID NO: 33)
CCCTCACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: 34)
GGGACGGAACAGCTTTGAGGTGTGCGTGAGGG 32-mer control (89-90 site): (SEQ
ID NO: 35) CCCTGCCTTGTCGAAACTCCACACGCACTCCC (SEQ ID NO: 36)
GGGAGTGCGTGTGGAGTTTCGACAAGGCAGGG 32-mer targeted (93-94 site): (SEQ
ID NO: 37) CCCUUCUGUCUUGAACAUGAGTTTTTTATGGC (SEQ ID NO: 38)
GCCATAAAAAACTCATGTTCAAGACAGAAGGG 32-mer control (93-94 site): (SEQ
ID NO: 39) CGGTATTTTTTGAGTACAAGTTCTGTCTTCCC (SEQ ID NO: 40)
GGGAAGACAGAACTTGTACTCAAAAAATACCG 37-mer targeted (93-94 site): (SEQ
ID NO: 41) CCCTTCTGTCTTGAACATGAGTTTTTTATGGCGGGAG (SEQ ID NO: 42)
CTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGG 37-mer control (93-94 site):
(SEQ ID NO: 43) GAGGGCGGTATTTTTTGAGTACAAGTTCTGTCTTCCC (SEQ ID NO:
44) GGGAAGACAGAACTTGTACTCAAAAAATACCGCCCTC 21-mer targeted (89-90
site): (SEQ ID NO: 45) ACCUCAAAGCUGUUCCGUCTT (SEQ ID NO: 46)
GACGGAACAGCUUUGAGGUTT 21-mer targeted (93-94 site): (SEQ ID NO: 47)
CCCUUCUGUCUUGAACAUGTT (SEQ ID NO: 48) CAUGUUCAAGACAGAAGGGTT
Example 10
Enhanced Cellular Stability of Double-Stranded 2'-O-Methyl RNA
In this example, the single-stranded control oligomer was
transfected at 800 nM. Accumulation was observed in the nucleus at
6 hours post transfection, however by 25 hours the fluorescence of
the single-stranded oligomer had largely dissipated, indicating the
oligomer was no longer intact (Fisher, T., T. Terhorst, et al.
(1993). "Intracellular disposition and metabolism of
fluorescently-labeled unmodified and modified oligonucleotides
microinjected into mammalian cells." Nucl. Acids Res.
21:3857-3865). The relative fluorescence of fluorescently-labeled
oligomers transfected into A549 cells was observed to fit the
following pattern:
TABLE-US-00012 single-stranded (800 nM) double-stranded (100 nm) 6
h ++++ +++++ 25 h + +++++
The double-stranded oligomer duplex, wherein the second strand was
2'-O-methyl modified RNA, was transfected at 100 nM, and was also
clearly visible at 6 hours post transfection. However, in contrast
to the single-stranded oligomer, the double-stranded was still
largely intact in the nucleus at 24 hours, even though the
concentration transfected was 8-fold less, thereby demonstrating
that the 2'-O-methyl second strand stabilized the oligomer in the
cell.
The oligomers were all 2'-O--CH.sub.3 with a phosphodiester
backbone containing 6-carboxyfluorescein (6-FAM) tethered to the 5'
hydroxyl. The single-stranded control oligomer was transfected at
800 nM complexed with 4 ug/mL of LIPOFECTAMINE.TM. 2000, and the
double-stranded complex was transfected at 100 nM complexed with 1
ug/mL of LIPOFECTAMINE.TM. 2000.
Uptake of the single and double-stranded oligonucleotides was
measured by fluorescent microscopy using an inverted microscope
with an excitation wavelength of 494 nm and an emission wavelength
of 519 nm. Some aliquots of cells were also stained with Dead Red,
a fluorescent reagent that measures the integrity of the cell
membrane and thus distinguishes live cells from dead cells. This
reagent is excited at a wavelength of 528 nm and emits a red
fluorescence at 617 nm. The Dead Red stain was supplied as a
1000.times. stock solution and was diluted to 1.times. with
Opti-Mem. It was applied to the cells for 20 minutes in a
humidified CO.sub.2 incubator and then removed and replaced with
Opti-Mem. Any background fluorescence can be reduced by rinsing the
cells with Opti-Mem and replacing with either full growth media
(e.g., DMEM with supplements) or with fresh Opti-Mem before
fluorescence microscopy.
Fluorescent signal was seen accumulating in the nucleus at 6 hours
post transfection, however by 24 hours the single-stranded oligomer
has significantly dissipated, indicating the oligomer is no longer
intact. The double-stranded duplexes (wherein the second strand is
2'-O-methyl modified RNA with a 5' 6-FAM) was transfected at 100
nM, and was also clearly visible at 6 hours post transfection. In
contrast to the single-stranded oligomer, the double-stranded was
still largely intact in the nucleus at 24 hours, even though the
concentration transfected was 8-fold less. This experiment
demonstrates that the 2'-O-methyl second strand stabilizes the
duplex in the cell.
Example 11
Enhanced Stability in Cells and Accumulation in Cytoplasm of RNA
Hybridized to 2'-O-Methyl RNA
The fluorescence signal, corresponding to uptake of FITC-labeled
RNA and 2'-O-methyl modified RNA duplexes, was measured at 6 and 24
hours. RNA complexes were transfected in A549 cells with 100 nM
oligomer complexed with 2 ug/mL LIPOFECTAMINE.TM. 2000 as described
below. Cells were continuously transfected for 24 hours and
fluorescent uptake was assessed at 6 and 24 hours. Oligomers were
2'-O-methyl modified RNA with 5' 6-FAM (FITC-2'-O-Me), 19-mer RNA
with two deoxynucleotides on the 3' end with 5' 6-FAM (FITC-RNA) or
19-mer RNA with two deoxynucleotides on the 3' end (RNA) complexed.
At 6 hours, the FITC-2'-O-methyl duplexes show localization in the
nucleus and the FITC-2'-O-methyl/RNA and 2'-O-methyl/FITC-RNA
complexes show a more diffuse pattern of uptake (these
RNA/2'-O-methyl complexes are a substrate for the RISC complex and
are therefore retained in the cytoplasm where the RISC complex has
been reported to be active). At 24 hours, the FITC-2'-O-methyl/RNA
and 2'-O-methyl/FITC-RNA complexes were still visible in the cell,
whereas typically not even the single-stranded FITC-2'-O-- was
visible, even when transfected at significantly higher
concentrations, demonstrating that the 2'-O-methyl RNA protects the
RNA strand from degradation in the cell.
RNA oligomers having a phosphodiester backbone with 2'-O-methyl
nucleotides were synthesized using standard phosphoramidite
chemistry. Oligomers were purified by denaturing polyacrylamide gel
electrophoresis (PAGE). Purity of oligomers was confirmed by (PAGE)
and mass spectrometry. All oligomers were greater than 90% full
length, and mass data obtained was consistent with expected values.
Target-specific siRNA duplexes consisted of 21-nt sense and 21-nt
antisense strands with symmetric 2-nt 3' deoxy TT overhangs. 21-nt
RNAs were chemically synthesized using phosphoramidite chemistry.
For duplex preparation, sense- and antisense oligomers (each at 50
.mu.M) were combined in equal volumes in annealing buffer (30 mM
HEPES pH 7.0, 100 mM potassium acetate, and 2 mM magnesium
acetate), heat-denatured at 90.degree. C. for 1 min and annealed at
37.degree. C. for one hour. Duplexes were stored at 80.degree. C.
until used.
A549 cells (American Type Culture Collection #CCL-185) were
cultured at 37.degree. C. in Dulbecco's Modified Eagle Medium
(DMEM, Invitrogen Corporation, cat. no. 11960-044) supplemented
with 2 mM L-glutamine, 100 units/mL penicillin, 100 .mu.g/mL
streptomycin, and 10% fetal bovine serum (FBS). HeLa cells
(American Type Culture Collection #CCL-2) were cultured at
37.degree. C. in Minimal Essential Medium (MEM, Invitrogen
Corporation, cat. no. 10370-021) supplemented with 2 mM
L-glutamine, 1.5 g/L sodium bicarbonate, 1.0 mM sodium pyruvate,
100 units/mL penicillin, 100 .mu.g/mL streptomycin, and 10% FBS.
Cells were passaged regularly to maintain exponential growth. On
the day prior to transfection, cells were trypsinized, counted, and
seeded in 48-well plates at a density of 20.times.10.sup.3 cells
per well in 250 .mu.L fresh media. On the day of transfection cells
were typically 60-65% confluent. Transfection of siRNA duplexes and
oligomers was carried out using LIPOFECTAMINE.TM. 2000 (Invitrogen
Corporation). Briefly, a 10.times. stock of LIPOFECTAMINE.TM. 2000
was prepared in Opti-Mem (Invitrogen Corporation) and incubated at
room temperature for 15 minutes. An equal volume of a 10.times.
stock of siRNA duplex or oligomers in Opti-Mem was added and
complexation carried out for 15 minutes at room temperature.
Complexes were then diluted 5-fold in full growth media. Culture
media was removed from each well prior to the addition of 250 .mu.L
complexes per well. Cells were incubated at 37.degree. C./5%
CO.sub.2 for 6 or 24 hours prior to assessing the uptake.
The results of this experiment, as well as the experiment set forth
in Example 10 demonstrate that the uptake of the double-stranded
oligonucleotides can be measured through the use of a detectable
label on either strand or on both strands.
Example 12
Protocol for Transfection of NHAC Cells
NHAC cells are obtained from Clonetics (San Diego, Calif. 92123).
On the day prior to transfection, approximately 4.5.times.10.sup.3
NHAC cells are plated in CGM (an NHAC cell culture media obtained
from Clontech) in each well of a 24-well plate. At the time of
transfection, the cells are preferably between about 70-80%
confluent. A 30 .mu.g/ml, 10.times. stock of LIPOFECTAMINE.TM. 2000
in Opti-Mem, a serum-free medium is prepared. This solution is
allowed to stand at room temperature for at least 15 minutes prior
to use. STEALTH.TM. RNA molecules which are 25 nucleotides in
length are used to transfect NHAC cells are prepared as a 3 .mu.M,
10.times. stock in Opti-Mem. Equal volumes of the
10.times.LIPOFECTAMINE.TM. 2000 and 10.times. STEALTH.TM. RNA
oligonucleotide stocks are then added and allowed the mixture to
incubate for 15 minutes to allow for complexation. This mixture is
then diluted with 4 volumes of CGM medium (Clontech) to form the
final 1.times. (300 nM STEALTH.TM. RNA oligonucleotide complexed
with 3 .mu.g/ml LIPOFECTAMINE.TM. 2000) solution for
transfection.
Prior to transfection, all media is aspirated from the NHAC cells
growing in the 24-well plate. 0.5 ml of the 1.times.
lipid/STEALTH.TM. RNA complex is added to each well, being careful
not to let the cells dry out during the change of media. The cells
are then incubated for 16-24 hours at 37.degree. C. in a humidified
CO.sub.2 incubator. The cells are then harvested, centrifuged to
obtain a cell pellet and analyzed for protein determination or RNA
isolation.
Example 13
Protocol for Transfection of THP-1 Cells by Electroporation
THP-1 cells are maintained in RPMI 1640 media containing 4.5 g/L
glucose, 10% FBS, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 10
mM HEPES, 1 mM sodium pyruvate, 5.times.10.sup.-5M
2-mercaptoethanol and Pen/Strep. The cells are split 1:3 twice a
week to maintain a cell concentration of 1-2.times.10.sup.6
cells/ml.
On the day prior to electroporation, THP-1 cells are seeded at
0.5.times.10.sup.6/ml. The cells are collected by centrifugation on
the next day and the cell pellet washed in PB-sucrose (70 ml 0.1M
sodium phosphate buffer, 272 ml of 1 M sucrose, 1 ml of 1M
MgCl.sub.2 and 657 ml water using sterile and endotoxin-free
solutions). 15 ml of PB-sucrose is used for every 50 ml of culture
that is started with. The cells are again centrifuged and re-washed
with PB-sucrose. The cells are then counted and centrifuged again.
The cell pellet is resuspended in PB-sucrose at a concentration of
15.times.10.sup.6 cells/ml.
For electroporation, 5 .mu.l of a 1 mM stock of STEALTH.TM. RNA
oligonucleotide is added to a sterile tube. To this 100 .mu.l of
the finally resuspended THP-1 cells is added and mixed gently. The
cells and STEALTH.TM. RNA oligonucleotide are then transferred to a
sterile 0.1 cm gap electroporation cuvette and electroporated using
a BioRad GENEPULSERII.TM. with RF module at a setting of: 100
volts, 100% modulation, 25 kHz frequency, 2 msec burst
duration.times.10 bursts with a burst interval of 100 msec.
Following electroporation, the cuvette is tapped to suspend any
settled cells and applied on additional burst at the above
settings. The cuvette is again taped gently immediately after the
pulse to avoid any pH gradients and 1.5 ml of warm RPMI 164 media
(described above) was added. The cells are then transferred into 2
wells of a 24-well plate (0.7 ml/well) for recovery. The cells are
incubated at 37.degree. C. for 24 hours, pelleted, and rinsed with
Opti-Mem to remove any excess STEALTH.TM. RNA oligonucleotide. The
cells can then be examined for STEALTH.TM. RNA oligonucleotide
uptake (if the STEALTH.TM. RNA oligonucleotide comprises a
detectable moiety) and can be lysed for RNA isolation or protein
determination.
Example 14
Protocol for Transfection of B6 and C3H Endothelial Cells
B6 or C3H cells are grown to confluence in either a 96- or 48-well
plate. This is typically achieved by plating B6 cells at
2.times.10.sup.4 cells/well in 50 .mu.l of DMEM media (5 ml FBS, 45
mg heparin, 6 ml of 5 mg/ml ECGF, 5 ml of Antibiotic-Antimycotic
(Invitrogen, Carlsbad, Calif.) and DMEM to a final volume of 500
ml) for 96 well plates or 6.times.10.sup.4 cells/well in 250 .mu.l
of DMEM media for 48 well plates. C3H cells are initially plated at
3.times.10.sup.4 cells/well in 50 .mu.l DMEM media for 96
well-plates or 6.times.10.sup.4 cells/well in 250 .mu.l of DMEM
media for 48 well plates. Initial platings are done 2 days prior to
transfection.
On the day of transfection, EPEI is diluted to a final
concentration of 5 .mu.M in water to create a 20.times. stock. A 40
.mu.g/ml 20.times. stock of LIPOFECTAMINE.TM. 2000 is also made by
mixing 80 .mu.l of LIPOFECTAMINE.TM. 2000 with 2 liters of OptiMem.
A 2 .mu.M, 10.times. stock of a STEALTH.TM. RNA oligonucleotide is
prepared in OptiMem. Each of the stock solutions is allowed to sit
at room temperature for 15 minutes. 42.5 .mu.l each of the EPEI and
LIPOFECTAMINE.TM. 2000 stocks is then mixed with 85 .mu.l of
STEALTH.TM. RNA oligonucleotide stock and allowed them to complex
for 15 minutes at room temperature. After complexing, the mixture
is diluted by adding 680 .mu.l of Opti-Mem. 50 .mu.l of the
complexed STEALTH.TM. RNA oligonucleotide is then added to each
well containing either B6 or C3H cells and incubated the cells for
2-3 hours at 37.degree. C. 5% CO.sub.2. The media is then aspirated
off and 50 or 250 .mu.l (depending upon whether the cells are in
48- or 96-well plates) of DMEM medium containing 1% FBS is added
and the cells are incubated overnight.
The following day, the cells are stimulated by the addition of 2
.mu.g/ml LPS in 1% FBS for 6 hours. Following LPS stimulation, the
cells can be lysed for RNA extraction and protein
determination.
Example 15
Exemplary Product Literature of the Invention
The text of exemplary product literature for various embodiments of
double-stranded oligonucleotides of the invention are disclosed
below. This product literature contains descriptions of kits of the
invention and includes instructions for use of kits of the
invention, and exemplary kit components. This exemplary product
literature is particularly useful for practicing various aspects of
business methods of the invention. Suitable methods and
compositions of the invention are described in the product
literature for the BLOCK-iT.TM. Fluorescent Oligo, BLOCK-iT.TM.
Transfection Optimization Kit, and Stealth.TM. RNA, all of which
are available from Invitrogen Corporation, Carlsbad, Calif.
BLOCK-iT.TM. Fluorescent Oligo
The BLOCK-iT.TM. Fluorescent Oligo is a fluorescein isothiocyanate
(FITC)-labeled dsRNA oligomer designed for use in RNAi analysis to
facilitate assessment and optimization of cationic lipid-mediated
delivery or electroporation of dsRNA oligonucleotides into
mammalian cells. Using the BLOCK-iT.TM. Fluorescent Oligo in RNAi
studies offers the following advantages: (1) provides a good
indication of the transfection efficiency with Invitrogen's
Stealth.TM. RNA, standard unmodified siRNA, or purified
Dicer-generated siRNA; and (2) allows strong, easy
fluorescence-based indication of transfection efficiency in every
RNAi experiment. The BLOCK-iT.TM. Fluorescent Oligo is supplied as
a 20 .mu.M or 1 mM stock of FITC-labeled double-stranded RNA
(dsRNA) oligomer in 100 mM KOAc, 30 mM HEPES-KOH, pH 7.4, and 2 mM
MgOAc. Upon receipt, the BLOCK-iT.TM. Fluorescent Oligo should be
stored at -20.degree. C., and protected from light.
The BLOCK-iT.TM. Fluorescent Oligo is a FITC-labeled,
double-stranded RNA duplex with the same length, charge, and
configuration as standard siRNA, and contains chemical
modifications that enhance the stability and allow assessment of
fluorescence signal for a significantly longer time period than is
obtained with other unmodified, fluorescently labeled RNA. For
example, fluorescence signal is readily detectable in HEK293 cells
for at least 72 hours. Note that the strength of the fluorescence
signal depends on the transfection efficiency, growth rate of the
cells, and the amount of oligomer transfected. The sequence of the
BLOCK-iT.TM. Fluorescent Oligo is not homologous to any known gene,
ensuring against induction of non-specific cellular events caused
by introduction of the Oligo into cells. The Oligo also localizes
primarily to the nucleus upon uptake (Fisher et al. 1993. Nuc.
Acids Res. 21:3857-3865). Importantly, the BLOCK-iT.TM. Fluorescent
Oligo is designed strictly for use as a tool for Stealth.TM. RNA or
siRNA uptake assessment, and is not meant to provide any
information about the behavior of a Stealth.TM. RNA or siRNA,
including its cellular localization, half-life, or stability.
The BLOCK-iT.TM. Fluorescent Oligo is supplied as a 20 .mu.M or 1
mM stock solution in an annealing buffer. The guidelines below
should be followed when handling the BLOCK-iT.TM. Fluorescent Oligo
stock solution: 1. The BLOCK-iT.TM. Fluorescent Oligo is light
sensitive. Store the stock solution at -20.degree. C., protected
from light. The stock solution is stable for at least 6 months if
stored properly. 2. When using, thaw the stock solution on ice or
at room temperature. Once thawed, place the tube on ice until use.
After use, return stock solution to -20.degree. C. storage. 3. The
stock solution may be frozen and thawed multiple times without loss
of fluorescence signal if handled properly. 4. Take precautions to
ensure that the stock solution does not become contaminated with
RNase. a. Use RNase-free sterile pipette tips and supplies for all
manipulations. b. Wear gloves when handling reagents and
solutions.
The BLOCK-iT.TM. Fluorescent Oligo (20 .mu.M or 1 mM stock) may be
used with any cationic lipid-based transfection reagent suitable
for delivery of Stealth.TM. RNA, siRNA, and Dicer-generated siRNA
to mammalian cells. For example, Lipofectamine.TM. 2000 Reagent
(Invitrogen Corp., Carlsbad, Calif.) provides for highly efficient
transfection of a wide variety of mammalian cells with the
BLOCK-iT.TM. Fluorescent Oligo (Ciccarone et al. 1999. Focus
21:54-55). The guidelines below should be followed when
transfecting the BLOCK-iT.TM. Fluorescent Oligo: 1. The amount of
BLOCK-iT.TM. Fluorescent Oligo to use depends on the growth rate
and transfection efficiency of the mammalian cells. When
transfecting a mammalian cell line for the first time, evaluate
several concentrations of lipid and vary the final concentration of
the BLOCK-iT.TM. Fluorescent Oligo from 10 to 200 nM to determine
the optimal amount of BLOCK-iT.TM. Fluorescent Oligo to use to
obtain a strong fluorescence signal. For most cell lines tested
(e.g. HEK293, A549, HeLa), w a readily detectable fluorescence
signal was obtained when using 100 nM BLOCK-iT.TM. Fluorescent
Oligo for transfection. 2. Prepare and seed mammalian cells at a
density recommended by the manufacturer of the transfection reagent
being used. 3. Prepare lipid-BLOCK-iT.TM. Fluorescent Oligo
complexes as directed by the manufacturer of the transfection
reagent being used. Always dilute the BLOCK-iT.TM. Fluorescent
Oligo immediately before transfection (i.e. do not store diluted
Oligo) and into an appropriate medium. For example, the
BLOCK-iT.TM. Fluorescent Oligo may be diluted into Opti-MEM.RTM. I
Reduced Serum Medium (Invitrogen Corp., Carlsbad, Calif.). 4.
Assess fluorescent uptake at 6 to 24 hours post-transfection. The
fluorescence signal may be detected at longer time points depending
on the transfection efficiency and growth rate of the cells.
When performing electroporation, higher concentrations of
BLOCK-iT.TM. Fluorescent Oligo may be required. Use the 1 mM stock
solution of BLOCK-iT.TM. Fluorescent Oligo to optimize
electroporation conditions according to the manufacturer's
guidelines.
Once the mammalian cells have been transfected or electroporated
with the BLOCK-iT.TM. Fluorescent Oligo, Oligo uptake in live cells
may be qualitatively assessed using fluorescence microscopy. Any
type of fluorescence microscope and any standard FITC filter set
(.lamda..sub.ex=494 nm, .lamda..sub.em=519 nm green) may be used
for detection.
BLOCK-iT.TM. Transfection Optimization Kit
The BLOCK-iT.TM. Transfection Optimization Kit is designed to
facilitate optimization of transfection conditions for RNAi
studies. The kit provides the following reagents: 1. BLOCK-iT.TM.
Fluorescent Oligo (20 .mu.M FITC-labeled double-stranded RNA
(dsRNA) oligomer in annealing buffer) for use as an indicator of
transfection efficiency in RNAi experiments with Stealth.TM. RNA or
siRNA. 2. A Stealth.TM. RNA molecule (20 .mu.M p53 Positive Control
Stealth.TM. RNA in annealing buffer) targeting the human p53 gene
for use as a positive control (in human cell lines only) for the
RNAi response. 3. A Scrambled Stealth.TM. RNA molecule (20 .mu.M
Scrambled Negative Control Stealth.TM. RNA in annealing buffer) for
use as a negative control (in human cell lines only) for the RNAi
response. 4. Dead Cell Reagent (2 mM Ethidium homodimer-1 (EthD-1)
in DMSO/H.sub.2O 1:4 (v/v)) to assess cell viability.
The annealing buffer is composed of 100 mM KOAc, 30 mM HEPES-KOH,
pH 7.4, and 2 mM MgOAc. Upon receipt, each of the above reagents
should be stored at -20.degree. C., and the BLOCK-iT.TM.
Fluorescent Oligo and Dead Cell Reagent should be protected from
light. All reagents in stock solutions are stable for at least 6
months when stored properly.
The BLOCK-iT.TM. Fluorescent Oligo and the Dead Cell Reagent in the
BLOCK-iT.TM. Transfection Optimization Kit can be used in RNAi
studies to help optimize transfection conditions for transfecting
Stealth.TM. RNA or siRNA into a mammalian cell line of interest for
the first time. The BLOCK-iT.TM. Fluorescent Oligo allows strong,
easy fluorescence-based assessment of dsRNA oligomer uptake into
mammalian cells. The BLOCK-iT.TM. Fluorescent Oligo is functionally
qualified by transient transfection into mammalian cells and
assessment of fluorescence signal at 24 hours
post-transfection.
Dead Cell Reagent is intended for use as an indicator of cell
viability following transfection of mammalian cells with
Stealth.TM. RNA or siRNA, and is an ethidium dye (ethidium
homodimer-1; EthD-1) with the following characteristics: (1)
Molecular formula: C.sub.46H.sub.50Cl.sub.4N.sub.8; and (2)
Molecular weight: 856.77. Dead Cell Reagent, which is excluded by
the intact plasma membrane of live cells, enters cells with damaged
membranes and emits a red fluorescence signal upon binding to
nucleic acids (.lamda..sub.ex=528 nm, .lamda..sub.em=617 nm). The
fluorescence signal is detectable using a fluorescence microscope
and filters for propidium iodide or Texas Red.RTM.. Once the
optimal conditions to use for transfection of a given cell line
have been determined, the BLOCK-iT.TM. Fluorescent Oligo and the
Dead Cell Reagent may be used in every RNAi experiment with that
cell line as an indicator of transfection efficiency and cell
viability.
Stealth.TM. RNAi is chemically modified dsRNA developed to overcome
the limitations of traditional siRNA. Using Stealth.TM. RNA for
RNAi analysis offers the following advantages: (1) obtain effective
target gene knockdown at levels that are equivalent to or greater
than those achieved with traditional siRNA; (2) reduces
non-specific effects caused by induction of cellular stress
response pathways; and (3) exhibits enhanced stability for greater
flexibility in RNAi analysis. The BLOCK-iT.TM. Transfection
Optimization Kit includes a p53 and a Scrambled Stealth.TM. RNA
molecule for use as positive and negative controls, respectively,
in an RNAi experiment targeting the human p53 gene. If the
mammalian cell line of interest is a human cell line that expresses
p53, the p53 and Scrambled Stealth.TM. RNA oligomers may be used as
positive and negative controls for the RNAi response, and to help
optimize transfection conditions. The p53 and Scrambled Stealth.TM.
RNA oligomers are functionally qualified by transient transfection
into A549 cells. At 24 hours post-transfection, mRNA is isolated
from treated and untreated cells using the mRNA Catcher.TM. Kit,
and qRT-PCR is performed using LUX.TM. primers for the human p53
gene. qRT-PCR analysis must demonstrate >75% inhibition of human
p53 expression levels in p53 Stealth.TM. RNA-treated cells and no
inhibition in Scrambled Stealth.TM. RNA-treated cells.
To properly handle the reagents of the BLOCK-iT.TM. Transfection
Optimization Kit, the stock solutions of the BLOCK-iT.TM.
Fluorescent Oligo and the Stealth.TM. RNA molecules should be
thawed on ice or at room temperature. Once thawed, the tubes should
be placed on ice until use. After use, the stock solution should be
returned to -20.degree. C. storage. The stock solution may be
frozen and thawed multiple times without loss of fluorescence
signal (BLOCK-iT.TM. Fluorescent Oligo) or activity (Control
Stealth.TM. RNAi oligomers) if handled properly. Precautions must
be taken when working with these reagents to ensure that the stock
solutions do not become contaminated with RNase. For example,
RNase-free sterile pipette tips and supplies should be used for all
manipulations, and gloves worn when handling the reagents. To
properly handle the Dead Cell Reagent, the stock solution should be
thawed at room temperature. To mix the stock solution, tap the
tube, and centrifuge briefly before opening. After use, return
stock solution to -20.degree. C. storage. The stock solution may be
frozen and thawed multiple times without loss of fluorescence
signal if handled properly.
Any suitable cationic lipid-based transfection reagent may be used
to deliver Stealth.TM. RNA or siRNA to mammalian cells. General
guidelines are provided below for using the reagents supplied in
the BLOCK-iT.TM. Transfection Optimization Kit to help optimize
transfection conditions for Stealth.TM. RNA or siRNA and mammalian
cell lines. For example, the Lipofectamine.TM. 2000 Reagent
(Invitrogen Corp., Carlsbad, Calif.) is optimal for highly
efficient delivery of Stealth.TM. RNA or siRNA to a wide variety of
mammalian cells (Gitlin et al., 2002. Nature 418:430-34; Yu et al.,
2002. Proc. Natl. Acad. Sci. USA 99:6047-6052). To perform a
transfection, first determine the appropriate amount of each
reagent to use such that fluorescence signal (BLOCK-iT.TM.
Fluorescent Oligo or Dead Cell Reagent) or gene knockdown effect
(p53 Stealth.TM. RNA) is readily detectable.
The amount of BLOCK-iT.TM. Fluorescent Oligo used to transfect a
mammalian cell line depends on the growth rate and transfection
efficiency of the mammalian cells. To optimize transfection
conditions, evaluate several concentrations of lipid and vary the
final concentration of the BLOCK-iT.TM. Fluorescent Oligo from 10
to 200 nM to determine the optimal amount of Oligo required to
obtain a strong fluorescence signal. A concentration of 100 nM
BLOCK-iT.TM. Fluorescent Oligo is a recommended starting point.
The amount of p53 Stealth.TM. RNA to transfect to achieve optimal
gene knockdown needs to be determined experimentally for each human
cell line. To optimize transfection conditions, evaluate several
concentrations of lipid and vary the final concentration of
Stealth.TM. RNA from 10 to 100 nM to determine the conditions
required for the optimal levels of gene knockdown. Use of higher
concentrations of Stealth.TM. RNA may be possible depending on the
cell line. A concentration of 40 nM p53 Stealth.TM. RNA is a
recommended starting point. The same concentration of the negative
control Scrambled Stealth.TM. RNA should be used.
The transfection experiment may be set up to allow for simultaneous
assessment of transfection efficiency and cell viability with the
same sample by transfecting one set of cells with the BLOCK-iT.TM.
Fluorescent Oligo, then staining those cells with the Dead Cell
Reagent at a suitable time period after transfection (generally 6
to 24 hours post-transfection). Prepare and seed cells at a density
recommended by the manufacturer of the transfection reagent being
used. Also prepare lipid-oligomer complexes as directed by the
manufacturer of the transfection reagent being used. The
BLOCK-iT.TM. Fluorescent Oligo or Stealth.TM. RNA should be diluted
immediately before transfection (i.e. do not store diluted
oligomer) and into an appropriate medium, for example,
Opti-MEM.RTM. I Reduced Serum Medium (Invitrogen Corp., Carlsbad,
Calif.).
The following procedure may be used to stain cells with Dead Cell
Reagent. First, prepare a sufficient amount of the working solution
based on the number of samples that will be stained. For example, 2
mls/well of staining solution should be prepared for a 6-well
culture vessel; 1 ml/well of staining solution should be prepared
for a 12-well culture vessel; 0.5 ml/well of staining solution
should be prepared for a 24-well culture vessel; and 0.25 ml/well
of staining solution should be prepared for a 48-well culture
vessel. To prepare the working solution, thaw the 2 mM Dead Cell
Reagent stock solution at room temperature. Tap the tube to mix,
and centrifuge briefly before opening. Dilute the appropriate
amount of Dead Cell Reagent into Opti-MEM.RTM. I Reduced Serum
Medium to prepare a 2 .mu.M working solution (1:1000 dilution). For
example, to prepare 1 ml of a 2 .mu.M working solution, add 1 .mu.l
of Dead Cell Reagent to 1 ml of Opti-MEM.RTM. I Reduced Serum
Medium. Aspirate the media from the cells and replace with the
appropriate volume of Dead Cell Reagent. Incubate the cells at
37.degree. C. in a CO.sub.2 incubator for 10-15 minutes, and then
remove the Dead Cell Reagent and replace with fresh Opti-MEM.RTM. I
Reduced Serum Medium.
After the mammalian cells have been transfected with the
BLOCK-iT.TM. Fluorescent Oligo and stained with Dead Cell Reagent,
Oligo uptake and cell viability may be qualitatively assessed using
any fluorescence microscope and the following filter sets. To
assess transfection efficiency, use any standard FITC filter set
(.lamda..sub.ex=494 nm, .lamda..sub.em=519 green) to detect the
fluorescence signal from the BLOCK-iT.TM. Fluorescent Oligo. To
assess cell viability, use a filter set for propidium iodide or
Texas Red.RTM. (.lamda..sub.ex=528 nm, .lamda..sub.em=617 nm) to
detect the fluorescence signal from the Dead Cell Reagent.
When the positive control Stealth.TM. RNA and the negative control
scrambled Stealth.TM. RNA are transfected into a mammalian cell
line, any method of choice may be used to detect human p53
expression levels. One exemplary method for assaying p53 mRNA
levels is quantitative RT-PCR (qRT-PCR) using Invitrogen's custom
LUX.TM. primers. The LUX.TM. Designer available at
www.invitrogen.com/lux may be used to help design and order
suitable primers to use for the qRT-PCR analysis. Invitrogen's mRNA
Catcher.TM. Kit may be used to prepare mRNA from treated or
untreated cells. When performing qRT-PCR, an internal control RNA
(e.g. .beta.-actin, GAPDH, or cyclophilin) should be used to
normalize results. Alternatively or in addition, Western blot
analysis using a suitable antibody to human p53 may be used to
assay for p53 protein levels. The half-life of the protein should
be taken into account when assessing RNAi effects at the protein
level.
Transfecting Stealth.TM. RNA or siRNA into Mammalian Cells Using
Lipofectamine.TM. 2000
As described above, Lipofectamine.TM. 2000 Reagent is a proprietary
formulation that facilitates highly efficient delivery of
Stealth.TM. RNA molecules or short interfering RNA (siRNA) to
mammalian cells for RNAi analysis. Below are general guidelines and
a procedure to transfect Stealth.TM. RNA or siRNA oligomers into
mammalian cells using Lipofectamine.TM. 2000. Recommended reagent
amounts are provided as a starting point. Transfection conditions
for the mammalian cell line and target gene used should be
optimized as described above for best results.
Many factors can influence the degree to which expression of a gene
of interest is reduced (i.e. gene knockdown) in an RNAi experiment
including, but not limited to, transfection efficiency,
transcription rate of the gene of interest, protein stability,
efficacy of the particular Stealth.TM. RNAi or siRNA sequence
chosen, and growth characteristics of the mammalian cell line. Take
these factors into account when designing transfection and RNAi
experiments. For more tips to help achieve success in RNAi
experiments, refer to the section below entitled "Seven Steps to
RNAi Success."
The following general guidelines will help optimize success when
transfecting Stealth.TM. RNA or siRNA into mammalian cells using
Lipofectamine.TM. 2000. Preferably the mammalian cell line of
interest used in the transfection experiments consists of
low-passage cells that are healthy, with greater than 90% viable
before transfection. The amount of Stealth.TM. RNA molecules or
siRNA to transfect to achieve optimal gene knockdown needs to be
determined experimentally for each cell line. The Stealth.TM. RNA
molecules or siRNA of interest may be suspended in annealing buffer
at a concentration of 20 .mu.M. For example, the BLOCK-iT.TM.
Fluorescent Oligo may be used to help optimize transfection
conditions for a cell line as described above. Once the optimal
conditions to use for transfection have been determined, the
BLOCK-iT.TM. Fluorescent Oligo may be included in every experiment
as an indicator of transfection efficiency.
If the mammalian cell line is being transfected for the first time,
evaluate several concentrations of Lipofectamine.TM. 2000 and vary
the final concentration of Stealth.TM. RNA or siRNA from 20 to 100
nM to determine the conditions required to achieve the optimal
levels of gene knockdown. Transfecting 40 nM of Stealth.TM. RNA or
siRNA is a good starting point. Higher concentrations of
Stealth.TM. RNA or siRNA may be possible depending on the cell
line. Transfect cells at 30-50% confluence. Gene knockdown levels
are generally assayed at a minimum of 24 to 72 hours following
transfection. Transfecting cells at a lower density allows a longer
interval between transfection and assay time, and minimizes the
loss of cell viability due to cell overgrowth. Depending on the
nature of the target gene, transfecting cells at higher densities
may be suitable with optimization of conditions. Do not add
antibiotics to the medium during transfection as this reduces
transfection efficiency and causes cell death. Finally, for optimal
results, use Opti-MEM.RTM. I Reduced Serum Medium (pre-warm to
37.degree. C. before use) to dilute Lipofectamine.TM. 2000 and
Stealth.TM. RNA or siRNA oligomers prior to complex formation.
The following procedure may be used to transfect Stealth.TM. RNA or
siRNA oligomers into mammalian cells using Lipofectamine.TM. 2000.
The Table below shows appropriate reagent amounts and volumes to
add for different tissue culture formats. Use the recommended
amounts of Stealth.TM. RNA or siRNA (see column 4) and
Lipofectamine.TM. 2000 (see column 6) as a starting point, and
optimize conditions for the cell line and Stealth.TM. RNA or siRNA
of interest (Note: 20 .mu.M Stealth.TM. RNA or siRNA=20
pmol/.mu.l).
TABLE-US-00013 Relative Stealth .TM. RNA Surface Volume Stealth
.TM. RNA or or siRNA Lipofectamine .TM. Lipofectamine .TM. Area of
siRNA (pmol) and Amounts 2000 (.mu.l) and 2000 Amounts Culture (vs.
Plating Dilution Volume (pmol) for Dilution Volume (.mu.l) for
Vessel 24-well) Medium (.mu.l) Optimization (.mu.l) Optimization
48-well 0.4 200 .mu.l 10 pmol in 25 .mu.l 2-25 pmol 0.5 .mu.l in 25
.mu.l 0.3-0.8 .mu.l 24-well 1 500 .mu.l 20 pmol in 50 .mu.l 10-50
pmol 1 .mu.l in 50 .mu.l 0.5-1.5 .mu.l 6-well 5 2 ml 100 pmol in
250 .mu.l 50-250 pmol 5 .mu.l in 250 .mu.l 2.5-6 .mu.l
To begin, one day before transfection, plate cells in the
appropriate amount of growth medium without antibiotics such that
they will be 30-50% confluent at the time of transfection. For each
transfection sample, prepare oligomer-Lipofectamine.TM. 2000
complexes as follows: (1) dilute Stealth.TM. RNA or siRNA oligomer
in the appropriate amount of Opti-MEM.RTM. I Reduced Serum Medium
without serum and mix gently; (2) mix Lipofectamine.TM. 2000 gently
before use, dilute the appropriate amount in Opti-MEM.RTM. I
Reduced Serum Medium, mix gently and incubate for 5 minutes at room
temperature; (3) after the 5-minute incubation, combine the diluted
oligomer with the diluted Lipofectamine.TM. 2000, mix gently and
incubate for 20 minutes at room temperature to allow complex
formation to occur. Note that the solution may appear cloudy, but
this will not inhibit transfection. Add the
oligomer-Lipofectamine.TM. 2000 complexes to each well containing
cells and medium. Mix gently by rocking the plate back and forth.
Finally, incubate the cells at 37.degree. C. in a CO.sub.2
incubator for 24-96 hours as appropriate and then assay for gene
knockdown. It is not necessary to remove the complexes or change
the medium; however, growth medium may be replaced after 4-6 hours
without loss of transfection activity.
The Table below shows optimal reagent amounts and volumes for a
variety of cell lines evaluated:
TABLE-US-00014 Opt. Duplex Optimal Lipid Duplex Lipid Cell Line
Species Type Description Conc. Conc. Range Range A549 human
adherent lung carcinoma 100 nM 2 ug/ml L2K HeLa human adherent
cervical 50 Nm 1 ug/ml L2K adenocarcinoma MC3T3 mouse adherent
fibroblast 1-100 nM 1-3 ug/ml Lipofectamine 2000 (L2K) 3T3-L1 mouse
adherent fibroblast 100 nM 2 ug/ml (Undifferentiated) L2K NRK rat
adherent normal rat 200 nM 2 ug/ml kidney L2K RAT 1 rat adherent
fibroblast 200 nM 2 ug/ml L2K HUVEC human adherent endothelial 300
nM 1:125 dilution of Oligofectamine HMVEC human adherent
microvascularendothelial 200 nM 2 ug/ml L2K HEK 293 human adherent
fetal kidney 200 nM 2 u/lml L2K HepG2 human adherent hepatocyte 300
nM 2 ug/ml L2K (while plating) MSC human adherent Mesenchymal 400
nM 2 ug/ml L2K 100-400 nM stem cells SK-N-SH human adherent
neuroblastoma 300 nM 3 ug/ml L2k Keratinocytes human adherent
primary 100 nM 2 ug/ml 10-200 nM 2-3 ug/ml keratinocytes L2K L2K
Sebocytes human adherent primary 100 Nm 3 ug/ml sebocytes L2K
Melanocytes human adherent primary 0.25-1 ug 1-3 u/l Mirus
melanocytes duplex/ TKO/ug well duplex (Mirus, Madison, WI, Cat No.
MIR 2154) HCT 116 human adherent colorectal 400 nM 4 ug/ml 200-800
nM 2-4 ug/ml carcinoma HNAC human adherent cartilage 300 nM 3 ug/ml
L2K C2C12 mouse adherent myoblast 50 nM 2 ug/ml L2K Primary mouse
adherent endothelial 200 nM 2 ug/ml L2K endothelium 0.25 uM EPEI
MCF7 human adherent breast 500 nM 3 ug/ml adenocarcinoma L2K (while
plating) RAW mouse adherent osteoclast 150 nM 2 ug/ml L2K 75-300 nM
264.7 Jurkat human suspension acute t-cell 50 uM. Electroporation
leukemia 100 volts 50% modulation 25 Khz 2 msec 10 bursts THP-1
human suspension acute monocytic 50 uM Electroporation leukemia 100
volts 100% modulation 25 Khz 2 msec 10 bursts Hut-78 human
suspension human t-cell 50 uM Electroporation 100 volts 80% n
modulation 25 Khz 2 msec 10 bursts
Seven Steps Toward RNAi Success
1. Optimize transfection conditions before beginning experiments
with Stealth.TM. RNA.
The level of confluence and passage number of the cells at the
start of transfection can have a significant impact on the
efficiency of Stealth.TM. RNA uptake and on the cellular toxicity
associated with transfection. Before beginning RNAi analysis,
optimize transfection conditions by determining the optimal cell
density and oligomer-lipid concentrations to use for the mammalian
cell line of interest and system. When optimizing transfection
conditions, follow these guidelines: (1) To ensure uniform uptake
of Stealth.TM. RNA, make sure that cells are plated uniformly
across the wells; (2) For highly efficient transfection in a broad
range of mammalian cell types, use Lipofectamine.TM. 2000 Reagent;
(3) Use the BLOCK-iT.TM. Fluorescent Oligo to optimize transfection
conditions as described above. Uptake of the BLOCK-iT.TM.
Fluorescent Oligo correlates strongly with uptake of Stealth.TM.
RNA or siRNA oligomers.
2. Include the BLOCK-iT.TM. Fluorescent Oligo in every
experiment.
The degree of the RNAi response to a particular Stealth.TM. RNA or
siRNA oligomer is directly linked to its transfection efficiency.
To assess transfection efficiency, include the BLOCK-iT.TM.
Fluorescent Oligo in every experiment. Using the BLOCK-iT.TM.
Fluorescent Oligo in transfection experiments allows for easy
assessment of oligomer uptake and transfection efficiency using any
fluorescence microscope and a standard FITC filter set. Uptake of
the fluorescent oligomer by at least 80% of cells correlates with
high levels of gene knockdown by effective Stealth.TM. RNA or siRNA
oligomers. Note that the BLOCK-iT.TM. Fluorescent Oligo is
chemically modified to enhance its stability and allows assessment
of fluorescence signal for a significantly longer time period than
is obtained with other unmodified, fluorescently-labeled RNA.
3. Assess Stealth.TM. RNAi or siRNA effects by performing an RNA
assay (i.e. qRT-PCR) first.
To validate Stealth.TM. RNA or siRNA oligomers, measure each
oligomer's effect on the target mRNA. Although many investigators
wish to bypass the RNA determination step and look directly at the
Stealth.TM. RNA or siRNA oligomer's effect on protein levels, this
is unadvisable, since the RNA assay will yield important
information about the rank order potency of the oligomers against
the target mRNA and provides valuable information required to
troubleshoot the assay system. For example, an RNAi oligomer may be
effective at decreasing mRNA levels of the target gene; however,
may not affect protein levels if the target protein has a long
half-life. Quantitative RT-PCR (qRT-PCR) using custom LUX.TM.
primers (Invitrogen Corp., Carlsbad, Calif.) provides a convenient
and high throughput method to evaluate the effect of an individual
or set of Stealth.TM. RNA or siRNA oligomers on target mRNA levels.
The LUX.TM. Designer available at www.invitrogen.com/lux may be
used to help design and order suitable primers for use in qRT-PCR
analysis. To prepare mRNA or total RNA from untreated or
oligomer-treated cells, mRNA Catcher.TM. Kit (Invitrogen Corp.,
Carlsbad, Calif.) or Concert.TM. 96 RNA Purification System
(Invitrogen Corp., Carlsbad, Calif.) may be used, respectively.
When performing qRT-PCR, results should be normalized to an
internal control RNA (e.g. .beta.-actin or GAPDH).
4. Know the half-life of the protein that you wish to inhibit.
To see Stealth.TM. RNA or siRNA-mediated inhibition at the protein
level, any pre-existing pool of the protein must be degraded. If
the protein of interest has a long half-life, long-term
transfection experiments may need to performed (i.e. perform
multiple cycles of transfection) to observe effects at the protein
level.
5. Always include the appropriate positive and negative
controls.
When performing RNAi analysis, it is important to include the
proper positive and negative controls to help evaluate experimental
results. For a positive control, include an effective Stealth.TM.
RNA for a target other than the mRNA of interest. For a negative
control, compare the levels of the target mRNA in Stealth.TM. RNA
or siRNA-treated and control (scrambled or reverse
sequences)-treated cells, for example by using the BLOCK-iT.TM.
Transfection Optimization Kit as described previously.
6. Follow these general guidelines to perform RNAi analysis using
Stealth.TM. RNA or siRNA.
When preparing oligomer-lipid complexes, dilute oligomer and lipid
into the appropriate medium, for example Opti-MEM.RTM. I Reduced
Serum Medium. Do not use phosphate-buffered saline (PBS) for
dilution, as transfection efficiency will be severely compromised.
Always mix the Stealth.TM. RNA or siRNA oligomer stock solution
thoroughly before use. Thaw, vortex, and spin to collect fluid
before removing sample. Do not allow the cells to dry out before
adding oligomer-lipid complexes. Doing so will reduce the
transfection efficiency and cell viability. For detailed protocols
to transfect Stealth.TM. RNA or siRNA oligomers, refer to the
manufacturer's instructions for the transfection reagent being
using.
7. Visit www.invitrogen.com/rnai for additional information,
resources, and protocols to help achieve RNAi analysis success.
Equivalents
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the following claims.
The entire contents of all patents, published patent applications
and other references cited herein are hereby expressly incorporated
herein in their entireties by reference.
SEQUENCE LISTINGS
1
49166DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 1ucuugaacau gaguugaaag aaaaactcat
gttcaagaca gaagggccga aagaaaggcc 60cuucug 66221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2cccuucuguc uugaacauga g 21321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 3ctgatgttca agacagaacg g 21421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4ctgatgttca agacagaacg g 21521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 5ctcauguuca agacagaagg g 21621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 6ctcauguuca agacagaagg g 21721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 7ctcauguuca agacagaagg g 21821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8gagtacaagt tctgtcttcc c 21921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9ggcaagacag aacttgtagt c 211021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10gggaagacag aacttgtact c 211121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 11gggaagacag aacttgtact c 211221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 12gggaagacag aacttgtact c 211358DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 13ggcccuucug ucuugaacau gaguugaaag aaaaactcat
gttcaagaca gaagggcc 581410PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 14His Ile Trp Leu Ile Tyr Leu
Trp Ile Val 1 5 101516PRTUnknown OrganismDescription of Unknown
Organism Antennapedia protein 15Arg Gln Ile Lys Ile Trp Phe Gln Asn
Arg Arg Met Lys Trp Lys Lys 1 5 10 151627PRTUnknown
OrganismDescription of Unknown Organism Transportan protein 16Gly
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10
15Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 251725PRTHuman
immunodeficiency virusMOD_RES(1)C(Acm) 17Cys Phe Ile Thr Lys Ala
Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg 1 5 10 15Arg Gln Arg Arg
Arg Pro Pro Gln Cys 20 251815PRTHuman immunodeficiency
virusMOD_RES(1)C(Acm) 18Cys Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
Pro Pro Gln Cys 1 5 10 151919PRTHuman immunodeficiency
virusMOD_RES(1)C(Acm) 19Cys Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg
Arg Gln Arg Arg Pro 1 5 10 15Pro Gln Cys2037PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Gly
Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg Lys Lys 1 5 10
15Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr His Gln Val
20 25 30Ser Leu Ser Lys Gln 352121DNAArtificial SequenceDescription
of Combined DNA/RNA Molecule Synthetic oligonucleotide 21accucaaagc
uguuccguct t 212221DNAArtificial SequenceDescription of Combined
DNA/RNA Molecule Synthetic oligonucleotide 22gacggaacag cuuugaggut
t 212327RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 23acgcacaccu caaagcuguu ccguccc
272427DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 24gggacggaac agctttgagg tgtgcgt
272527RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 25acgcacaccu caaagcuguu ccguccc
272627DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 26gggacggaac agctttgagg tgtgcgt
272727DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 27ccctgccttg tcgaaactcc acacgca
272827DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 28tgcgtgtgga gtttcgacaa ggcaggg
272932DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 29ccctcacgca caccucaaag
cuguuccguc cc 323032DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 30gggacggaac agctttgagg
tgtgcgtgag gg 323132DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 31ccctgccttg tcgaaactcc
acacgcactc cc 323232DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 32gggagtgcgt gtggagtttc
gacaaggcag gg 323332DNAArtificial SequenceDescription of Combined
DNA/RNA Molecule Synthetic oligonucleotide 33ccctcacgca caccucaaag
cuguuccguc cc 323432DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 34gggacggaac agctttgagg
tgtgcgtgag gg 323532DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 35ccctgccttg tcgaaactcc
acacgcactc cc 323632DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 36gggagtgcgt gtggagtttc
gacaaggcag gg 323732DNAArtificial SequenceDescription of Combined
DNA/RNA Molecule Synthetic oligonucleotide 37cccuucuguc uugaacauga
gttttttatg gc 323832DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 38gccataaaaa actcatgttc
aagacagaag gg 323932DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 39cggtattttt tgagtacaag
ttctgtcttc cc 324032DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 40gggaagacag aacttgtact
caaaaaatac cg 324137DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 41cccttctgtc ttgaacatga
gttttttatg gcgggag 374237DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 42ctcccgccat
aaaaaactca tgttcaagac agaaggg 374337DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 43gagggcggta ttttttgagt acaagttctg tcttccc
374437DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 44gggaagacag aacttgtact caaaaaatac
cgccctc 374521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 45accucaaagc uguuccguct t
214621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 46gacggaacag cuuugaggut t
214721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 47cccuucuguc uugaacaugt t
214821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 48cauguucaag acagaagggt t
21494PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Asp Glu Val Asp 1
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References